Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

PubMed

Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

PubMed Central

Bergman, Casey M.; Haddrill, Penelope R.

2015-01-01

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center. PMID:25717372

Strain-specific and pooled genome sequences for populations of Drosophila melanogaster from three continents.

PubMed

Bergman, Casey M; Haddrill, Penelope R

2015-01-01

To contribute to our general understanding of the evolutionary forces that shape variation in genome sequences in nature, we have sequenced genomes from 50 isofemale lines and six pooled samples from populations of Drosophila melanogaster on three continents. Analysis of raw and reference-mapped reads indicates the quality of these genomic sequence data is very high. Comparison of the predicted and experimentally-determined Wolbachia infection status of these samples suggests that strain or sample swaps are unlikely to have occurred in the generation of these data. Genome sequences are freely available in the European Nucleotide Archive under accession ERP009059. Isofemale lines can be obtained from the Drosophila Species Stock Center.

HUGO: Hierarchical mUlti-reference Genome cOmpression for aligned reads

PubMed Central

Li, Pinghao; Jiang, Xiaoqian; Wang, Shuang; Kim, Jihoon; Xiong, Hongkai; Ohno-Machado, Lucila

2014-01-01

Background and objective Short-read sequencing is becoming the standard of practice for the study of structural variants associated with disease. However, with the growth of sequence data largely surpassing reasonable storage capability, the biomedical community is challenged with the management, transfer, archiving, and storage of sequence data. Methods We developed Hierarchical mUlti-reference Genome cOmpression (HUGO), a novel compression algorithm for aligned reads in the sorted Sequence Alignment/Map (SAM) format. We first aligned short reads against a reference genome and stored exactly mapped reads for compression. For the inexact mapped or unmapped reads, we realigned them against different reference genomes using an adaptive scheme by gradually shortening the read length. Regarding the base quality value, we offer lossy and lossless compression mechanisms. The lossy compression mechanism for the base quality values uses k-means clustering, where a user can adjust the balance between decompression quality and compression rate. The lossless compression can be produced by setting k (the number of clusters) to the number of different quality values. Results The proposed method produced a compression ratio in the range 0.5–0.65, which corresponds to 35–50% storage savings based on experimental datasets. The proposed approach achieved 15% more storage savings over CRAM and comparable compression ratio with Samcomp (CRAM and Samcomp are two of the state-of-the-art genome compression algorithms). The software is freely available at https://sourceforge.net/projects/hierachicaldnac/with a General Public License (GPL) license. Limitation Our method requires having different reference genomes and prolongs the execution time for additional alignments. Conclusions The proposed multi-reference-based compression algorithm for aligned reads outperforms existing single-reference based algorithms. PMID:24368726

Geoseq: a tool for dissecting deep-sequencing datasets.

PubMed

Gurtowski, James; Cancio, Anthony; Shah, Hardik; Levovitz, Chaya; George, Ajish; Homann, Robert; Sachidanandam, Ravi

2010-10-12

Datasets generated on deep-sequencing platforms have been deposited in various public repositories such as the Gene Expression Omnibus (GEO), Sequence Read Archive (SRA) hosted by the NCBI, or the DNA Data Bank of Japan (ddbj). Despite being rich data sources, they have not been used much due to the difficulty in locating and analyzing datasets of interest. Geoseq http://geoseq.mssm.edu provides a new method of analyzing short reads from deep sequencing experiments. Instead of mapping the reads to reference genomes or sequences, Geoseq maps a reference sequence against the sequencing data. It is web-based, and holds pre-computed data from public libraries. The analysis reduces the input sequence to tiles and measures the coverage of each tile in a sequence library through the use of suffix arrays. The user can upload custom target sequences or use gene/miRNA names for the search and get back results as plots and spreadsheet files. Geoseq organizes the public sequencing data using a controlled vocabulary, allowing identification of relevant libraries by organism, tissue and type of experiment. Analysis of small sets of sequences against deep-sequencing datasets, as well as identification of public datasets of interest, is simplified by Geoseq. We applied Geoseq to, a) identify differential isoform expression in mRNA-seq datasets, b) identify miRNAs (microRNAs) in libraries, and identify mature and star sequences in miRNAS and c) to identify potentially mis-annotated miRNAs. The ease of using Geoseq for these analyses suggests its utility and uniqueness as an analysis tool.

Whole Transcriptome Sequencing Enables Discovery and Analysis of Viruses in Archived Primary Central Nervous System Lymphomas

PubMed Central

DeBoever, Christopher; Reid, Erin G.; Smith, Erin N.; Wang, Xiaoyun; Dumaop, Wilmar; Harismendy, Olivier; Carson, Dennis; Richman, Douglas; Masliah, Eliezer; Frazer, Kelly A.

2013-01-01

Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples. PMID:24023918

2. Photocopy of photograph (from Reading Co. Archives) Photographer unknown ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. Photocopy of photograph (from Reading Co. Archives) Photographer unknown ca. 1937 NORTHEAST FRONT AND SOUTHEAST SIDE - Philadelphia & Reading Railroad, Terminal Station, 1115-1141 Market Street, Philadelphia, Philadelphia County, PA

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

PubMed

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives

PubMed Central

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information. PMID:28234924

Non-synonymous variations in cancer and their effects on the human proteome: workflow for NGS data biocuration and proteome-wide analysis of TCGA data.

PubMed

Cole, Charles; Krampis, Konstantinos; Karagiannis, Konstantinos; Almeida, Jonas S; Faison, William J; Motwani, Mona; Wan, Quan; Golikov, Anton; Pan, Yang; Simonyan, Vahan; Mazumder, Raja

2014-01-27

Next-generation sequencing (NGS) technologies have resulted in petabytes of scattered data, decentralized in archives, databases and sometimes in isolated hard-disks which are inaccessible for browsing and analysis. It is expected that curated secondary databases will help organize some of this Big Data thereby allowing users better navigate, search and compute on it. To address the above challenge, we have implemented a NGS biocuration workflow and are analyzing short read sequences and associated metadata from cancer patients to better understand the human variome. Curation of variation and other related information from control (normal tissue) and case (tumor) samples will provide comprehensive background information that can be used in genomic medicine research and application studies. Our approach includes a CloudBioLinux Virtual Machine which is used upstream of an integrated High-performance Integrated Virtual Environment (HIVE) that encapsulates Curated Short Read archive (CSR) and a proteome-wide variation effect analysis tool (SNVDis). As a proof-of-concept, we have curated and analyzed control and case breast cancer datasets from the NCI cancer genomics program - The Cancer Genome Atlas (TCGA). Our efforts include reviewing and recording in CSR available clinical information on patients, mapping of the reads to the reference followed by identification of non-synonymous Single Nucleotide Variations (nsSNVs) and integrating the data with tools that allow analysis of effect nsSNVs on the human proteome. Furthermore, we have also developed a novel phylogenetic analysis algorithm that uses SNV positions and can be used to classify the patient population. The workflow described here lays the foundation for analysis of short read sequence data to identify rare and novel SNVs that are not present in dbSNP and therefore provides a more comprehensive understanding of the human variome. Variation results for single genes as well as the entire study are available from the CSR website (http://hive.biochemistry.gwu.edu/dna.cgi?cmd=csr). Availability of thousands of sequenced samples from patients provides a rich repository of sequence information that can be utilized to identify individual level SNVs and their effect on the human proteome beyond what the dbSNP database provides.

Non-synonymous variations in cancer and their effects on the human proteome: workflow for NGS data biocuration and proteome-wide analysis of TCGA data

PubMed Central

2014-01-01

Background Next-generation sequencing (NGS) technologies have resulted in petabytes of scattered data, decentralized in archives, databases and sometimes in isolated hard-disks which are inaccessible for browsing and analysis. It is expected that curated secondary databases will help organize some of this Big Data thereby allowing users better navigate, search and compute on it. Results To address the above challenge, we have implemented a NGS biocuration workflow and are analyzing short read sequences and associated metadata from cancer patients to better understand the human variome. Curation of variation and other related information from control (normal tissue) and case (tumor) samples will provide comprehensive background information that can be used in genomic medicine research and application studies. Our approach includes a CloudBioLinux Virtual Machine which is used upstream of an integrated High-performance Integrated Virtual Environment (HIVE) that encapsulates Curated Short Read archive (CSR) and a proteome-wide variation effect analysis tool (SNVDis). As a proof-of-concept, we have curated and analyzed control and case breast cancer datasets from the NCI cancer genomics program - The Cancer Genome Atlas (TCGA). Our efforts include reviewing and recording in CSR available clinical information on patients, mapping of the reads to the reference followed by identification of non-synonymous Single Nucleotide Variations (nsSNVs) and integrating the data with tools that allow analysis of effect nsSNVs on the human proteome. Furthermore, we have also developed a novel phylogenetic analysis algorithm that uses SNV positions and can be used to classify the patient population. The workflow described here lays the foundation for analysis of short read sequence data to identify rare and novel SNVs that are not present in dbSNP and therefore provides a more comprehensive understanding of the human variome. Variation results for single genes as well as the entire study are available from the CSR website (http://hive.biochemistry.gwu.edu/dna.cgi?cmd=csr). Conclusions Availability of thousands of sequenced samples from patients provides a rich repository of sequence information that can be utilized to identify individual level SNVs and their effect on the human proteome beyond what the dbSNP database provides. PMID:24467687

EBI metagenomics--a new resource for the analysis and archiving of metagenomic data.

PubMed

Hunter, Sarah; Corbett, Matthew; Denise, Hubert; Fraser, Matthew; Gonzalez-Beltran, Alejandra; Hunter, Christopher; Jones, Philip; Leinonen, Rasko; McAnulla, Craig; Maguire, Eamonn; Maslen, John; Mitchell, Alex; Nuka, Gift; Oisel, Arnaud; Pesseat, Sebastien; Radhakrishnan, Rajesh; Rocca-Serra, Philippe; Scheremetjew, Maxim; Sterk, Peter; Vaughan, Daniel; Cochrane, Guy; Field, Dawn; Sansone, Susanna-Assunta

2014-01-01

Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive.

EBI metagenomics—a new resource for the analysis and archiving of metagenomic data

PubMed Central

Hunter, Sarah; Corbett, Matthew; Denise, Hubert; Fraser, Matthew; Gonzalez-Beltran, Alejandra; Hunter, Christopher; Jones, Philip; Leinonen, Rasko; McAnulla, Craig; Maguire, Eamonn; Maslen, John; Mitchell, Alex; Nuka, Gift; Oisel, Arnaud; Pesseat, Sebastien; Radhakrishnan, Rajesh; Rocca-Serra, Philippe; Scheremetjew, Maxim; Sterk, Peter; Vaughan, Daniel; Cochrane, Guy; Field, Dawn; Sansone, Susanna-Assunta

2014-01-01

Metagenomics is a relatively recently established but rapidly expanding field that uses high-throughput next-generation sequencing technologies to characterize the microbial communities inhabiting different ecosystems (including oceans, lakes, soil, tundra, plants and body sites). Metagenomics brings with it a number of challenges, including the management, analysis, storage and sharing of data. In response to these challenges, we have developed a new metagenomics resource (http://www.ebi.ac.uk/metagenomics/) that allows users to easily submit raw nucleotide reads for functional and taxonomic analysis by a state-of-the-art pipeline, and have them automatically stored (together with descriptive, standards-compliant metadata) in the European Nucleotide Archive. PMID:24165880

TagDigger: user-friendly extraction of read counts from GBS and RAD-seq data.

PubMed

Clark, Lindsay V; Sacks, Erik J

2016-01-01

In genotyping-by-sequencing (GBS) and restriction site-associated DNA sequencing (RAD-seq), read depth is important for assessing the quality of genotype calls and estimating allele dosage in polyploids. However, existing pipelines for GBS and RAD-seq do not provide read counts in formats that are both accurate and easy to access. Additionally, although existing pipelines allow previously-mined SNPs to be genotyped on new samples, they do not allow the user to manually specify a subset of loci to examine. Pipelines that do not use a reference genome assign arbitrary names to SNPs, making meta-analysis across projects difficult. We created the software TagDigger, which includes three programs for analyzing GBS and RAD-seq data. The first script, tagdigger_interactive.py, rapidly extracts read counts and genotypes from FASTQ files using user-supplied sets of barcodes and tags. Input and output is in CSV format so that it can be opened by spreadsheet software. Tag sequences can also be imported from the Stacks, TASSEL-GBSv2, TASSEL-UNEAK, or pyRAD pipelines, and a separate file can be imported listing the names of markers to retain. A second script, tag_manager.py, consolidates marker names and sequences across multiple projects. A third script, barcode_splitter.py, assists with preparing FASTQ data for deposit in a public archive by splitting FASTQ files by barcode and generating MD5 checksums for the resulting files. TagDigger is open-source and freely available software written in Python 3. It uses a scalable, rapid search algorithm that can process over 100 million FASTQ reads per hour. TagDigger will run on a laptop with any operating system, does not consume hard drive space with intermediate files, and does not require programming skill to use.

Alignment of 1000 Genomes Project reads to reference assembly GRCh38.

PubMed

Zheng-Bradley, Xiangqun; Streeter, Ian; Fairley, Susan; Richardson, David; Clarke, Laura; Flicek, Paul

2017-07-01

The 1000 Genomes Project produced more than 100 trillion basepairs of short read sequence from more than 2600 samples in 26 populations over a period of five years. In its final phase, the project released over 85 million genotyped and phased variants on human reference genome assembly GRCh37. An updated reference assembly, GRCh38, was released in late 2013, but there was insufficient time for the final phase of the project analysis to change to the new assembly. Although it is possible to lift the coordinates of the 1000 Genomes Project variants to the new assembly, this is a potentially error-prone process as coordinate remapping is most appropriate only for non-repetitive regions of the genome and those that did not see significant change between the two assemblies. It will also miss variants in any region that was newly added to GRCh38. Thus, to produce the highest quality variants and genotypes on GRCh38, the best strategy is to realign the reads and recall the variants based on the new alignment. As the first step of variant calling for the 1000 Genomes Project data, we have finished remapping all of the 1000 Genomes sequence reads to GRCh38 with alternative scaffold-aware BWA-MEM. The resulting alignments are available as CRAM, a reference-based sequence compression format. The data have been released on our FTP site and are also available from European Nucleotide Archive to facilitate researchers discovering variants on the primary sequences and alternative contigs of GRCh38. © The Authors 2017. Published by Oxford University Press.

ExpEdit: a webserver to explore human RNA editing in RNA-Seq experiments.

PubMed

Picardi, Ernesto; D'Antonio, Mattia; Carrabino, Danilo; Castrignanò, Tiziana; Pesole, Graziano

2011-05-01

ExpEdit is a web application for assessing RNA editing in human at known or user-specified sites supported by transcript data obtained by RNA-Seq experiments. Mapping data (in SAM/BAM format) or directly sequence reads [in FASTQ/short read archive (SRA) format] can be provided as input to carry out a comparative analysis against a large collection of known editing sites collected in DARNED database as well as other user-provided potentially edited positions. Results are shown as dynamic tables containing University of California, Santa Cruz (UCSC) links for a quick examination of the genomic context. ExpEdit is freely available on the web at http://www.caspur.it/ExpEdit/.

SCALCE: boosting sequence compression algorithms using locally consistent encoding.

PubMed

Hach, Faraz; Numanagic, Ibrahim; Alkan, Can; Sahinalp, S Cenk

2012-12-01

The high throughput sequencing (HTS) platforms generate unprecedented amounts of data that introduce challenges for the computational infrastructure. Data management, storage and analysis have become major logistical obstacles for those adopting the new platforms. The requirement for large investment for this purpose almost signalled the end of the Sequence Read Archive hosted at the National Center for Biotechnology Information (NCBI), which holds most of the sequence data generated world wide. Currently, most HTS data are compressed through general purpose algorithms such as gzip. These algorithms are not designed for compressing data generated by the HTS platforms; for example, they do not take advantage of the specific nature of genomic sequence data, that is, limited alphabet size and high similarity among reads. Fast and efficient compression algorithms designed specifically for HTS data should be able to address some of the issues in data management, storage and communication. Such algorithms would also help with analysis provided they offer additional capabilities such as random access to any read and indexing for efficient sequence similarity search. Here we present SCALCE, a 'boosting' scheme based on Locally Consistent Parsing technique, which reorganizes the reads in a way that results in a higher compression speed and compression rate, independent of the compression algorithm in use and without using a reference genome. Our tests indicate that SCALCE can improve the compression rate achieved through gzip by a factor of 4.19-when the goal is to compress the reads alone. In fact, on SCALCE reordered reads, gzip running time can improve by a factor of 15.06 on a standard PC with a single core and 6 GB memory. Interestingly even the running time of SCALCE + gzip improves that of gzip alone by a factor of 2.09. When compared with the recently published BEETL, which aims to sort the (inverted) reads in lexicographic order for improving bzip2, SCALCE + gzip provides up to 2.01 times better compression while improving the running time by a factor of 5.17. SCALCE also provides the option to compress the quality scores as well as the read names, in addition to the reads themselves. This is achieved by compressing the quality scores through order-3 Arithmetic Coding (AC) and the read names through gzip through the reordering SCALCE provides on the reads. This way, in comparison with gzip compression of the unordered FASTQ files (including reads, read names and quality scores), SCALCE (together with gzip and arithmetic encoding) can provide up to 3.34 improvement in the compression rate and 1.26 improvement in running time. Our algorithm, SCALCE (Sequence Compression Algorithm using Locally Consistent Encoding), is implemented in C++ with both gzip and bzip2 compression options. It also supports multithreading when gzip option is selected, and the pigz binary is available. It is available at http://scalce.sourceforge.net. fhach@cs.sfu.ca or cenk@cs.sfu.ca Supplementary data are available at Bioinformatics online.

NABIC: A New Access Portal to Search, Visualize, and Share Agricultural Genomics Data.

PubMed

Seol, Young-Joo; Lee, Tae-Ho; Park, Dong-Suk; Kim, Chang-Kug

2016-01-01

The National Agricultural Biotechnology Information Center developed an access portal to search, visualize, and share agricultural genomics data with a focus on South Korean information and resources. The portal features an agricultural biotechnology database containing a wide range of omics data from public and proprietary sources. We collected 28.4 TB of data from 162 agricultural organisms, with 10 types of omics data comprising next-generation sequencing sequence read archive, genome, gene, nucleotide, DNA chip, expressed sequence tag, interactome, protein structure, molecular marker, and single-nucleotide polymorphism datasets. Our genomic resources contain information on five animals, seven plants, and one fungus, which is accessed through a genome browser. We also developed a data submission and analysis system as a web service, with easy-to-use functions and cutting-edge algorithms, including those for handling next-generation sequencing data.

Masking as an effective quality control method for next-generation sequencing data analysis.

PubMed

Yun, Sajung; Yun, Sijung

2014-12-13

Next generation sequencing produces base calls with low quality scores that can affect the accuracy of identifying simple nucleotide variation calls, including single nucleotide polymorphisms and small insertions and deletions. Here we compare the effectiveness of two data preprocessing methods, masking and trimming, and the accuracy of simple nucleotide variation calls on whole-genome sequence data from Caenorhabditis elegans. Masking substitutes low quality base calls with 'N's (undetermined bases), whereas trimming removes low quality bases that results in a shorter read lengths. We demonstrate that masking is more effective than trimming in reducing the false-positive rate in single nucleotide polymorphism (SNP) calling. However, both of the preprocessing methods did not affect the false-negative rate in SNP calling with statistical significance compared to the data analysis without preprocessing. False-positive rate and false-negative rate for small insertions and deletions did not show differences between masking and trimming. We recommend masking over trimming as a more effective preprocessing method for next generation sequencing data analysis since masking reduces the false-positive rate in SNP calling without sacrificing the false-negative rate although trimming is more commonly used currently in the field. The perl script for masking is available at http://code.google.com/p/subn/. The sequencing data used in the study were deposited in the Sequence Read Archive (SRX450968 and SRX451773).

Quark enables semi-reference-based compression of RNA-seq data.

PubMed

Sarkar, Hirak; Patro, Rob

2017-11-01

The past decade has seen an exponential increase in biological sequencing capacity, and there has been a simultaneous effort to help organize and archive some of the vast quantities of sequencing data that are being generated. Although these developments are tremendous from the perspective of maximizing the scientific utility of available data, they come with heavy costs. The storage and transmission of such vast amounts of sequencing data is expensive. We present Quark, a semi-reference-based compression tool designed for RNA-seq data. Quark makes use of a reference sequence when encoding reads, but produces a representation that can be decoded independently, without the need for a reference. This allows Quark to achieve markedly better compression rates than existing reference-free schemes, while still relieving the burden of assuming a specific, shared reference sequence between the encoder and decoder. We demonstrate that Quark achieves state-of-the-art compression rates, and that, typically, only a small fraction of the reference sequence must be encoded along with the reads to allow reference-free decompression. Quark is implemented in C ++11, and is available under a GPLv3 license at www.github.com/COMBINE-lab/quark. rob.patro@cs.stonybrook.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

SCALCE: boosting sequence compression algorithms using locally consistent encoding

PubMed Central

Hach, Faraz; Numanagić, Ibrahim; Sahinalp, S Cenk

2012-01-01

Motivation: The high throughput sequencing (HTS) platforms generate unprecedented amounts of data that introduce challenges for the computational infrastructure. Data management, storage and analysis have become major logistical obstacles for those adopting the new platforms. The requirement for large investment for this purpose almost signalled the end of the Sequence Read Archive hosted at the National Center for Biotechnology Information (NCBI), which holds most of the sequence data generated world wide. Currently, most HTS data are compressed through general purpose algorithms such as gzip. These algorithms are not designed for compressing data generated by the HTS platforms; for example, they do not take advantage of the specific nature of genomic sequence data, that is, limited alphabet size and high similarity among reads. Fast and efficient compression algorithms designed specifically for HTS data should be able to address some of the issues in data management, storage and communication. Such algorithms would also help with analysis provided they offer additional capabilities such as random access to any read and indexing for efficient sequence similarity search. Here we present SCALCE, a ‘boosting’ scheme based on Locally Consistent Parsing technique, which reorganizes the reads in a way that results in a higher compression speed and compression rate, independent of the compression algorithm in use and without using a reference genome. Results: Our tests indicate that SCALCE can improve the compression rate achieved through gzip by a factor of 4.19—when the goal is to compress the reads alone. In fact, on SCALCE reordered reads, gzip running time can improve by a factor of 15.06 on a standard PC with a single core and 6 GB memory. Interestingly even the running time of SCALCE + gzip improves that of gzip alone by a factor of 2.09. When compared with the recently published BEETL, which aims to sort the (inverted) reads in lexicographic order for improving bzip2, SCALCE + gzip provides up to 2.01 times better compression while improving the running time by a factor of 5.17. SCALCE also provides the option to compress the quality scores as well as the read names, in addition to the reads themselves. This is achieved by compressing the quality scores through order-3 Arithmetic Coding (AC) and the read names through gzip through the reordering SCALCE provides on the reads. This way, in comparison with gzip compression of the unordered FASTQ files (including reads, read names and quality scores), SCALCE (together with gzip and arithmetic encoding) can provide up to 3.34 improvement in the compression rate and 1.26 improvement in running time. Availability: Our algorithm, SCALCE (Sequence Compression Algorithm using Locally Consistent Encoding), is implemented in C++ with both gzip and bzip2 compression options. It also supports multithreading when gzip option is selected, and the pigz binary is available. It is available at http://scalce.sourceforge.net. Contact: fhach@cs.sfu.ca or cenk@cs.sfu.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23047557

NABIC: A New Access Portal to Search, Visualize, and Share Agricultural Genomics Data

PubMed Central

Seol, Young-Joo; Lee, Tae-Ho; Park, Dong-Suk; Kim, Chang-Kug

2016-01-01

The National Agricultural Biotechnology Information Center developed an access portal to search, visualize, and share agricultural genomics data with a focus on South Korean information and resources. The portal features an agricultural biotechnology database containing a wide range of omics data from public and proprietary sources. We collected 28.4 TB of data from 162 agricultural organisms, with 10 types of omics data comprising next-generation sequencing sequence read archive, genome, gene, nucleotide, DNA chip, expressed sequence tag, interactome, protein structure, molecular marker, and single-nucleotide polymorphism datasets. Our genomic resources contain information on five animals, seven plants, and one fungus, which is accessed through a genome browser. We also developed a data submission and analysis system as a web service, with easy-to-use functions and cutting-edge algorithms, including those for handling next-generation sequencing data. PMID:26848255

Database resources of the National Center for Biotechnology Information.

PubMed

Sayers, Eric W; Barrett, Tanya; Benson, Dennis A; Bolton, Evan; Bryant, Stephen H; Canese, Kathi; Chetvernin, Vyacheslav; Church, Deanna M; Dicuccio, Michael; Federhen, Scott; Feolo, Michael; Fingerman, Ian M; Geer, Lewis Y; Helmberg, Wolfgang; Kapustin, Yuri; Krasnov, Sergey; Landsman, David; Lipman, David J; Lu, Zhiyong; Madden, Thomas L; Madej, Tom; Maglott, Donna R; Marchler-Bauer, Aron; Miller, Vadim; Karsch-Mizrachi, Ilene; Ostell, James; Panchenko, Anna; Phan, Lon; Pruitt, Kim D; Schuler, Gregory D; Sequeira, Edwin; Sherry, Stephen T; Shumway, Martin; Sirotkin, Karl; Slotta, Douglas; Souvorov, Alexandre; Starchenko, Grigory; Tatusova, Tatiana A; Wagner, Lukas; Wang, Yanli; Wilbur, W John; Yaschenko, Eugene; Ye, Jian

2012-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Website. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Genome and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, BioProject, BioSample, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Probe, Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), Biosystems, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information

PubMed Central

Acland, Abigail; Agarwala, Richa; Barrett, Tanya; Beck, Jeff; Benson, Dennis A.; Bollin, Colleen; Bolton, Evan; Bryant, Stephen H.; Canese, Kathi; Church, Deanna M.; Clark, Karen; DiCuccio, Michael; Dondoshansky, Ilya; Federhen, Scott; Feolo, Michael; Geer, Lewis Y.; Gorelenkov, Viatcheslav; Hoeppner, Marilu; Johnson, Mark; Kelly, Christopher; Khotomlianski, Viatcheslav; Kimchi, Avi; Kimelman, Michael; Kitts, Paul; Krasnov, Sergey; Kuznetsov, Anatoliy; Landsman, David; Lipman, David J.; Lu, Zhiyong; Madden, Thomas L.; Madej, Tom; Maglott, Donna R.; Marchler-Bauer, Aron; Karsch-Mizrachi, Ilene; Murphy, Terence; Ostell, James; O'Sullivan, Christopher; Panchenko, Anna; Phan, Lon; Pruitt, Don Preussm Kim D.; Rubinstein, Wendy; Sayers, Eric W.; Schneider, Valerie; Schuler, Gregory D.; Sequeira, Edwin; Sherry, Stephen T.; Shumway, Martin; Sirotkin, Karl; Siyan, Karanjit; Slotta, Douglas; Soboleva, Alexandra; Soussov, Vladimir; Starchenko, Grigory; Tatusova, Tatiana A.; Trawick, Bart W.; Vakatov, Denis; Wang, Yanli; Ward, Minghong; John Wilbur, W.; Yaschenko, Eugene; Zbicz, Kerry

2014-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, PubReader, Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link, Primer-BLAST, COBALT, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, the Genetic Testing Registry, Genome and related tools, the Map Viewer, Trace Archive, Sequence Read Archive, BioProject, BioSample, ClinVar, MedGen, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Probe, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool, Biosystems, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All these resources can be accessed through the NCBI home page. PMID:24259429

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

PubMed

Gautam, Aarti; Kumar, Raina; Dimitrov, George; Hoke, Allison; Hammamieh, Rasha; Jett, Marti

2016-10-01

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.

78 FR 47245 - NARA Records Subject to FOIA

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-05

... the NARA Web site, available at: http://www.archives.gov/foia/electronic-reading-room.html . (b) The... 31, 1996, also will be placed on NARA's Web site at http://www.archives.gov/foia/electronic-reading... you faster if we have any questions about your request. It is incumbent on the requester to maintain a...

Uncovering Information Hidden in Web Archives: Glimpse at Web Analysis Building on Data Warehouses; Towards Continuous Web Archiving: First Results and an Agenda for the Future; The Open Video Digital Library; After Migration to an Electronic Journal Collection: Impact on Faculty and Doctoral Students; Who Is Reading On-Line Education Journals? Why? And What Are They Reading?; Report on eLibrary@UBC4: Research, Collaboration and the Digital Library - Visions for 2010.

ERIC Educational Resources Information Center

Rauber, Andreas; Bruckner, Robert M.; Aschenbrenner, Andreas; Witvoet, Oliver; Kaiser, Max; Masanes, Julien; Marchionini, Gary; Geisler, Gary; King, Donald W.; Montgomery, Carol Hansen; Rudner, Lawrence M.; Gellmann, Jennifer S.; Miller-Whitehead, Marie; Iverson, Lee

2002-01-01

These six articles discuss Web archives and Web analysis building on data warehouses; international efforts at continuous Web archiving; the Open Video Digital Library; electronic journal collections in academic libraries; online education journals; and an electronic library symposium at the University of British Columbia. (LRW)

8. Photocopy of circa 1892 construction photograph, courtesy of Reading ...

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8. Photocopy of circa 1892 construction photograph, courtesy of Reading Company Archives - Philadelphia & Reading Railroad, Terminal Station, 1115-1141 Market Street, Philadelphia, Philadelphia County, PA

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Actionable mutations in canine hemangiosarcoma

PubMed Central

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A.; Wojcik, John; Durham, Amy C.; Mason, Nicola J.

2017-01-01

Background Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Methods Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Results and conclusions Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease. PMID:29190660

Actionable mutations in canine hemangiosarcoma.

PubMed

Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

2017-01-01

Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Interpretation of ANA Indirect Immunofluorescence Test Outside the Darkroom Using NOVA View Compared to Manual Microscopy

PubMed Central

Copple, Susan S.; Jaskowski, Troy D.; Giles, Rashelle; Hill, Harry R.

2014-01-01

Objective. To evaluate NOVA View with focus on reading archived images versus microscope based manual interpretation of ANA HEp-2 slides by an experienced, certified medical technologist. Methods. 369 well defined sera from: 44 rheumatoid arthritis, 50 systemic lupus erythematosus, 35 scleroderma, 19 Sjögren's syndrome, and 10 polymyositis patients as well as 99 healthy controls were examined. In addition, 12 defined sera from the Centers for Disease Control and 100 random patient sera sent to ARUP Laboratories for ANA HEp-2 IIF testing were included. Samples were read using the archived images on NOVA View and compared to results obtained from manual reading. Results. At a 1 : 40/1 : 80 dilution the resulting comparison demonstrated 94.8%/92.9% positive, 97.4%/97.4% negative, and 96.5%/96.2% total agreements between manual IIF and NOVA View archived images. Agreement of identifiable patterns between methods was 97%, with PCNA and mixed patterns undetermined. Conclusion. Excellent agreements were obtained between reading archived images on NOVA View and manually on a fluorescent microscope. In addition, workflow benefits were observed which need to be analyzed in future studies. PMID:24741573

Database resources of the National Center for Biotechnology Information

PubMed Central

Sayers, Eric W.; Barrett, Tanya; Benson, Dennis A.; Bolton, Evan; Bryant, Stephen H.; Canese, Kathi; Chetvernin, Vyacheslav; Church, Deanna M.; DiCuccio, Michael; Federhen, Scott; Feolo, Michael; Fingerman, Ian M.; Geer, Lewis Y.; Helmberg, Wolfgang; Kapustin, Yuri; Krasnov, Sergey; Landsman, David; Lipman, David J.; Lu, Zhiyong; Madden, Thomas L.; Madej, Tom; Maglott, Donna R.; Marchler-Bauer, Aron; Miller, Vadim; Karsch-Mizrachi, Ilene; Ostell, James; Panchenko, Anna; Phan, Lon; Pruitt, Kim D.; Schuler, Gregory D.; Sequeira, Edwin; Sherry, Stephen T.; Shumway, Martin; Sirotkin, Karl; Slotta, Douglas; Souvorov, Alexandre; Starchenko, Grigory; Tatusova, Tatiana A.; Wagner, Lukas; Wang, Yanli; Wilbur, W. John; Yaschenko, Eugene; Ye, Jian

2012-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Website. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Genome and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, BioProject, BioSample, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Probe, Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), Biosystems, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov. PMID:22140104

Database resources of the National Center for Biotechnology Information

PubMed Central

2013-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, the Genetic Testing Registry, Genome and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, BioProject, BioSample, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Probe, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool, Biosystems, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page. PMID:23193264

Database resources of the National Center for Biotechnology Information

PubMed Central

2015-01-01

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts for published life science journals. Additional NCBI resources focus on literature (Bookshelf, PubMed Central (PMC) and PubReader); medical genetics (ClinVar, dbMHC, the Genetic Testing Registry, HIV-1/Human Protein Interaction Database and MedGen); genes and genomics (BioProject, BioSample, dbSNP, dbVar, Epigenomics, Gene, Gene Expression Omnibus (GEO), Genome, HomoloGene, the Map Viewer, Nucleotide, PopSet, Probe, RefSeq, Sequence Read Archive, the Taxonomy Browser, Trace Archive and UniGene); and proteins and chemicals (Biosystems, COBALT, the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), the Molecular Modeling Database (MMDB), Protein Clusters, Protein and the PubChem suite of small molecule databases). The Entrez system provides search and retrieval operations for many of these databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at http://www.ncbi.nlm.nih.gov. PMID:25398906

Database resources of the National Center for Biotechnology Information

PubMed Central

2016-01-01

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts for published life science journals. Additional NCBI resources focus on literature (PubMed Central (PMC), Bookshelf and PubReader), health (ClinVar, dbGaP, dbMHC, the Genetic Testing Registry, HIV-1/Human Protein Interaction Database and MedGen), genomes (BioProject, Assembly, Genome, BioSample, dbSNP, dbVar, Epigenomics, the Map Viewer, Nucleotide, Probe, RefSeq, Sequence Read Archive, the Taxonomy Browser and the Trace Archive), genes (Gene, Gene Expression Omnibus (GEO), HomoloGene, PopSet and UniGene), proteins (Protein, the Conserved Domain Database (CDD), COBALT, Conserved Domain Architecture Retrieval Tool (CDART), the Molecular Modeling Database (MMDB) and Protein Clusters) and chemicals (Biosystems and the PubChem suite of small molecule databases). The Entrez system provides search and retrieval operations for most of these databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov. PMID:26615191

The BIG Data Center: from deposition to integration to translation

PubMed Central

2017-01-01

Biological data are generated at unprecedentedly exponential rates, posing considerable challenges in big data deposition, integration and translation. The BIG Data Center, established at Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, provides a suite of database resources, including (i) Genome Sequence Archive, a data repository specialized for archiving raw sequence reads, (ii) Gene Expression Nebulas, a data portal of gene expression profiles based entirely on RNA-Seq data, (iii) Genome Variation Map, a comprehensive collection of genome variations for featured species, (iv) Genome Warehouse, a centralized resource housing genome-scale data with particular focus on economically important animals and plants, (v) Methylation Bank, an integrated database of whole-genome single-base resolution methylomes and (vi) Science Wikis, a central access point for biological wikis developed for community annotations. The BIG Data Center is dedicated to constructing and maintaining biological databases through big data integration and value-added curation, conducting basic research to translate big data into big knowledge and providing freely open access to a variety of data resources in support of worldwide research activities in both academia and industry. All of these resources are publicly available and can be found at http://bigd.big.ac.cn. PMID:27899658

ENDEMIC INFECTION OF STRANDED SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS) WITH NOVEL PARVOVIRUS, POLYOMAVIRUS, AND ADENOVIRUS.

PubMed

Siqueira, Juliana D; Ng, Terry F; Miller, Melissa; Li, Linlin; Deng, Xutao; Dodd, Erin; Batac, Francesca; Delwart, Eric

2017-07-01

Over the past century, the southern sea otter (SSO; Enhydra lutris nereis) population has been slowly recovering from near extinction due to overharvest. The SSO is a threatened subspecies under federal law and a fully protected species under California law, US. Through a multiagency collaborative program, stranded animals are rehabilitated and released, while deceased animals are necropsied and tissues are cryopreserved to facilitate scientific study. Here, we processed archival tissues to enrich particle-associated viral nucleic acids, which we randomly amplified and deeply sequenced to identify viral genomes through sequence similarities. Anelloviruses and endogenous retroviral sequences made up over 50% of observed viral sequences. Polyomavirus, parvovirus, and adenovirus sequences made up most of the remaining reads. We characterized and phylogenetically analyzed the full genome of sea otter polyomavirus 1 and the complete coding sequence of sea otter parvovirus 1 and found that the closest known viruses infect primates and domestic pigs ( Sus scrofa domesticus), respectively. We tested archived tissues from 69 stranded SSO necropsied over 14 yr (2000-13) by PCR. Polyomavirus, parvovirus, and adenovirus infections were detected in 51, 61, and 29% of examined animals, respectively, with no significant increase in frequency over time, suggesting endemic infection. We found that 80% of tested SSO were infected with at least one of the three DNA viruses, whose tissue distribution we determined in 261 tissue samples. Parvovirus DNA was most frequently detected in mesenteric lymph node, polyomavirus DNA in spleen, and adenovirus DNA in multiple tissues (spleen, retropharyngeal and mesenteric lymph node, lung, and liver). This study describes the virome in tissues of a threatened species and shows that stranded SSO are frequently infected with multiple viruses, warranting future research to investigate associations between these infections and observed lesions.

Database Resources of the BIG Data Center in 2018

PubMed Central

Xu, Xingjian; Hao, Lili; Zhu, Junwei; Tang, Bixia; Zhou, Qing; Song, Fuhai; Chen, Tingting; Zhang, Sisi; Dong, Lili; Lan, Li; Wang, Yanqing; Sang, Jian; Hao, Lili; Liang, Fang; Cao, Jiabao; Liu, Fang; Liu, Lin; Wang, Fan; Ma, Yingke; Xu, Xingjian; Zhang, Lijuan; Chen, Meili; Tian, Dongmei; Li, Cuiping; Dong, Lili; Du, Zhenglin; Yuan, Na; Zeng, Jingyao; Zhang, Zhewen; Wang, Jinyue; Shi, Shuo; Zhang, Yadong; Pan, Mengyu; Tang, Bixia; Zou, Dong; Song, Shuhui; Sang, Jian; Xia, Lin; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Zhang, Yang; Sheng, Xin; Lu, Mingming; Wang, Qi; Xiao, Jingfa; Zou, Dong; Wang, Fan; Hao, Lili; Liang, Fang; Li, Mengwei; Sun, Shixiang; Zou, Dong; Li, Rujiao; Yu, Chunlei; Wang, Guangyu; Sang, Jian; Liu, Lin; Li, Mengwei; Li, Man; Niu, Guangyi; Cao, Jiabao; Sun, Shixiang; Xia, Lin; Yin, Hongyan; Zou, Dong; Xu, Xingjian; Ma, Lina; Chen, Huanxin; Sun, Yubin; Yu, Lei; Zhai, Shuang; Sun, Mingyuan; Zhang, Zhang; Zhao, Wenming; Xiao, Jingfa; Bao, Yiming; Song, Shuhui; Hao, Lili; Li, Rujiao; Ma, Lina; Sang, Jian; Wang, Yanqing; Tang, Bixia; Zou, Dong; Wang, Fan

2018-01-01

Abstract The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. PMID:29036542

AF Week in Photos > U.S. Air Force > Article Display

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Professors Senior Mentor Biographies Fact Sheets Commander's Call Topics CCT Archive CSAF Reading List 2017 CSAF Reading List 2016 CSAF Reading List 2015 CSAF Reading List 2014 CSAF Reading List 2013 CSAF Reading List 2012 CSAF Reading List 2011 CSAF Reading List 2010 CSAF Reading List 2009 CSAF Reading List

Workflow and web application for annotating NCBI BioProject transcriptome data

PubMed Central

Vera Alvarez, Roberto; Medeiros Vidal, Newton; Garzón-Martínez, Gina A.; Barrero, Luz S.; Landsman, David

2017-01-01

Abstract The volume of transcriptome data is growing exponentially due to rapid improvement of experimental technologies. In response, large central resources such as those of the National Center for Biotechnology Information (NCBI) are continually adapting their computational infrastructure to accommodate this large influx of data. New and specialized databases, such as Transcriptome Shotgun Assembly Sequence Database (TSA) and Sequence Read Archive (SRA), have been created to aid the development and expansion of centralized repositories. Although the central resource databases are under continual development, they do not include automatic pipelines to increase annotation of newly deposited data. Therefore, third-party applications are required to achieve that aim. Here, we present an automatic workflow and web application for the annotation of transcriptome data. The workflow creates secondary data such as sequencing reads and BLAST alignments, which are available through the web application. They are based on freely available bioinformatics tools and scripts developed in-house. The interactive web application provides a search engine and several browser utilities. Graphical views of transcript alignments are available through SeqViewer, an embedded tool developed by NCBI for viewing biological sequence data. The web application is tightly integrated with other NCBI web applications and tools to extend the functionality of data processing and interconnectivity. We present a case study for the species Physalis peruviana with data generated from BioProject ID 67621. Database URL: http://www.ncbi.nlm.nih.gov/projects/physalis/ PMID:28605765

High-throughput detection of RNA processing in bacteria.

PubMed

Gill, Erin E; Chan, Luisa S; Winsor, Geoffrey L; Dobson, Neil; Lo, Raymond; Ho Sui, Shannan J; Dhillon, Bhavjinder K; Taylor, Patrick K; Shrestha, Raunak; Spencer, Cory; Hancock, Robert E W; Unrau, Peter J; Brinkman, Fiona S L

2018-03-27

Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely - 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com .

Tidying Up International Nucleotide Sequence Databases: Ecological, Geographical and Sequence Quality Annotation of ITS Sequences of Mycorrhizal Fungi

PubMed Central

Tedersoo, Leho; Abarenkov, Kessy; Nilsson, R. Henrik; Schüssler, Arthur; Grelet, Gwen-Aëlle; Kohout, Petr; Oja, Jane; Bonito, Gregory M.; Veldre, Vilmar; Jairus, Teele; Ryberg, Martin; Larsson, Karl-Henrik; Kõljalg, Urmas

2011-01-01

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi. PMID:21949797

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment.

PubMed

Gierliński, Marek; Cole, Christian; Schofield, Pietà; Schurch, Nicholas J; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J

2015-11-15

High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of 'bad' replicates, which can drastically affect the gene read-count distribution. RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. g.j.barton@dundee.ac.uk. © The Author 2015. Published by Oxford University Press.

Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment

PubMed Central

Cole, Christian; Schofield, Pietà; Schurch, Nicholas J.; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J.

2015-01-01

Motivation: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read-count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. Results: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ∼0.01. The high-replicate data also allowed for strict quality control and screening of ‘bad’ replicates, which can drastically affect the gene read-count distribution. Availability and implementation: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. Contact: g.j.barton@dundee.ac.uk PMID:26206307

Read Across Texas! 2002 Texas Reading Club Manual.

ERIC Educational Resources Information Center

Edgmon, Missy; Ferate-Soto, Paolo; Foley, Lelana; Hager, Tina; Heard, Adriana; Ingham, Donna; Lopez, Nohemi; McMahon, Dorothy; Meyer, Sally; Parrish, Leila; Rodriguez-Gibbs, Josefina; Moreyra-Torres, Maricela; Travis, Gayle; Welch, Willy

The goal of the Texas Reading Club is to encourage the children of Texas to become library users and lifelong readers. This manual was created for the 2002 Texas Reading Club, a program of the Texas State Library and Archives Commission. The theme, "Read Across Texas!" invites children to explore the history, geography, and culture of…

Arkas: Rapid reproducible RNAseq analysis

PubMed Central

Colombo, Anthony R.; J. Triche Jr, Timothy; Ramsingh, Giridharan

2017-01-01

The recently introduced Kallisto pseudoaligner has radically simplified the quantification of transcripts in RNA-sequencing experiments.  We offer cloud-scale RNAseq pipelines Arkas-Quantification, and Arkas-Analysis available within Illumina’s BaseSpace cloud application platform which expedites Kallisto preparatory routines, reliably calculates differential expression, and performs gene-set enrichment of REACTOME pathways .  Due to inherit inefficiencies of scale, Illumina's BaseSpace computing platform offers a massively parallel distributive environment improving data management services and data importing.   Arkas-Quantification deploys Kallisto for parallel cloud computations and is conveniently integrated downstream from the BaseSpace Sequence Read Archive (SRA) import/conversion application titled SRA Import.  Arkas-Analysis annotates the Kallisto results by extracting structured information directly from source FASTA files with per-contig metadata, calculates the differential expression and gene-set enrichment analysis on both coding genes and transcripts. The Arkas cloud pipeline supports ENSEMBL transcriptomes and can be used downstream from the SRA Import facilitating raw sequencing importing, SRA FASTQ conversion, RNA quantification and analysis steps. PMID:28868134

NCBI Epigenomics: a new public resource for exploring epigenomic data sets

PubMed Central

Fingerman, Ian M.; McDaniel, Lee; Zhang, Xuan; Ratzat, Walter; Hassan, Tarek; Jiang, Zhifang; Cohen, Robert F.; Schuler, Gregory D.

2011-01-01

The Epigenomics database at the National Center for Biotechnology Information (NCBI) is a new resource that has been created to serve as a comprehensive public resource for whole-genome epigenetic data sets (www.ncbi.nlm.nih.gov/epigenomics). Epigenetics is the study of stable and heritable changes in gene expression that occur independently of the primary DNA sequence. Epigenetic mechanisms include post-translational modifications of histones, DNA methylation, chromatin conformation and non-coding RNAs. It has been observed that misregulation of epigenetic processes has been associated with human disease. We have constructed the new resource by selecting the subset of epigenetics-specific data from general-purpose archives, such as the Gene Expression Omnibus, and Sequence Read Archives, and then subjecting them to further review, annotation and reorganization. Raw data is processed and mapped to genomic coordinates to generate ‘tracks’ that are a visual representation of the data. These data tracks can be viewed using popular genome browsers or downloaded for local analysis. The Epigenomics resource also provides the user with a unique interface that allows for intuitive browsing and searching of data sets based on biological attributes. Currently, there are 69 studies, 337 samples and over 1100 data tracks from five well-studied species that are viewable and downloadable in Epigenomics. PMID:21075792

NCBI Epigenomics: a new public resource for exploring epigenomic data sets.

PubMed

Fingerman, Ian M; McDaniel, Lee; Zhang, Xuan; Ratzat, Walter; Hassan, Tarek; Jiang, Zhifang; Cohen, Robert F; Schuler, Gregory D

2011-01-01

The Epigenomics database at the National Center for Biotechnology Information (NCBI) is a new resource that has been created to serve as a comprehensive public resource for whole-genome epigenetic data sets (www.ncbi.nlm.nih.gov/epigenomics). Epigenetics is the study of stable and heritable changes in gene expression that occur independently of the primary DNA sequence. Epigenetic mechanisms include post-translational modifications of histones, DNA methylation, chromatin conformation and non-coding RNAs. It has been observed that misregulation of epigenetic processes has been associated with human disease. We have constructed the new resource by selecting the subset of epigenetics-specific data from general-purpose archives, such as the Gene Expression Omnibus, and Sequence Read Archives, and then subjecting them to further review, annotation and reorganization. Raw data is processed and mapped to genomic coordinates to generate 'tracks' that are a visual representation of the data. These data tracks can be viewed using popular genome browsers or downloaded for local analysis. The Epigenomics resource also provides the user with a unique interface that allows for intuitive browsing and searching of data sets based on biological attributes. Currently, there are 69 studies, 337 samples and over 1100 data tracks from five well-studied species that are viewable and downloadable in Epigenomics.

Database resources of the National Center for Biotechnology Information.

PubMed

Sayers, Eric W; Barrett, Tanya; Benson, Dennis A; Bolton, Evan; Bryant, Stephen H; Canese, Kathi; Chetvernin, Vyacheslav; Church, Deanna M; DiCuccio, Michael; Federhen, Scott; Feolo, Michael; Fingerman, Ian M; Geer, Lewis Y; Helmberg, Wolfgang; Kapustin, Yuri; Landsman, David; Lipman, David J; Lu, Zhiyong; Madden, Thomas L; Madej, Tom; Maglott, Donna R; Marchler-Bauer, Aron; Miller, Vadim; Mizrachi, Ilene; Ostell, James; Panchenko, Anna; Phan, Lon; Pruitt, Kim D; Schuler, Gregory D; Sequeira, Edwin; Sherry, Stephen T; Shumway, Martin; Sirotkin, Karl; Slotta, Douglas; Souvorov, Alexandre; Starchenko, Grigory; Tatusova, Tatiana A; Wagner, Lukas; Wang, Yanli; Wilbur, W John; Yaschenko, Eugene; Ye, Jian

2011-01-01

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Electronic PCR, OrfFinder, Splign, ProSplign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), IBIS, Biosystems, Peptidome, OMSSA, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

The BIG Data Center: from deposition to integration to translation.

PubMed

2017-01-04

Biological data are generated at unprecedentedly exponential rates, posing considerable challenges in big data deposition, integration and translation. The BIG Data Center, established at Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, provides a suite of database resources, including (i) Genome Sequence Archive, a data repository specialized for archiving raw sequence reads, (ii) Gene Expression Nebulas, a data portal of gene expression profiles based entirely on RNA-Seq data, (iii) Genome Variation Map, a comprehensive collection of genome variations for featured species, (iv) Genome Warehouse, a centralized resource housing genome-scale data with particular focus on economically important animals and plants, (v) Methylation Bank, an integrated database of whole-genome single-base resolution methylomes and (vi) Science Wikis, a central access point for biological wikis developed for community annotations. The BIG Data Center is dedicated to constructing and maintaining biological databases through big data integration and value-added curation, conducting basic research to translate big data into big knowledge and providing freely open access to a variety of data resources in support of worldwide research activities in both academia and industry. All of these resources are publicly available and can be found at http://bigd.big.ac.cn. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Database Resources of the BIG Data Center in 2018.

PubMed

2018-01-04

The BIG Data Center at Beijing Institute of Genomics (BIG) of the Chinese Academy of Sciences provides freely open access to a suite of database resources in support of worldwide research activities in both academia and industry. With the vast amounts of omics data generated at ever-greater scales and rates, the BIG Data Center is continually expanding, updating and enriching its core database resources through big-data integration and value-added curation, including BioCode (a repository archiving bioinformatics tool codes), BioProject (a biological project library), BioSample (a biological sample library), Genome Sequence Archive (GSA, a data repository for archiving raw sequence reads), Genome Warehouse (GWH, a centralized resource housing genome-scale data), Genome Variation Map (GVM, a public repository of genome variations), Gene Expression Nebulas (GEN, a database of gene expression profiles based on RNA-Seq data), Methylation Bank (MethBank, an integrated databank of DNA methylomes), and Science Wikis (a series of biological knowledge wikis for community annotations). In addition, three featured web services are provided, viz., BIG Search (search as a service; a scalable inter-domain text search engine), BIG SSO (single sign-on as a service; a user access control system to gain access to multiple independent systems with a single ID and password) and Gsub (submission as a service; a unified submission service for all relevant resources). All of these resources are publicly accessible through the home page of the BIG Data Center at http://bigd.big.ac.cn. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Next-Generation Sequencing of the Chrysanthemum nankingense (Asteraceae) Transcriptome Permits Large-Scale Unigene Assembly and SSR Marker Discovery

PubMed Central

Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Peng, Hui; Li, Pirui; Song, Aiping; Guan, Zhiyong; Fang, Weimin; Liao, Yuan; Chen, Fadi

2013-01-01

Background Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low. Methodology/Principal Findings Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity. Conclusions/Significance SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera. PMID:23626799

IMNGS: A comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies.

PubMed

Lagkouvardos, Ilias; Joseph, Divya; Kapfhammer, Martin; Giritli, Sabahattin; Horn, Matthias; Haller, Dirk; Clavel, Thomas

2016-09-23

The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.

A Longitudinal Study of Reading Comprehension Achievement from Grades 3 to 10: Investigating Models of Stability, Cumulative Growth, and Compensation

ERIC Educational Resources Information Center

Kwiatkowska-White, Bozena; Kirby, John R.; Lee, Elizabeth A.

2016-01-01

This longitudinal study of 78 Canadian English-speaking students examined the applicability of the stability, cumulative, and compensatory models in reading comprehension development. Archival government-mandated assessments of reading comprehension at Grades 3, 6, and 10, and the Canadian Test of Basic Skills measure of reading comprehension…

Sanger and Next-Generation Sequencing data for characterization of CTL epitopes in archived HIV-1 proviral DNA.

PubMed

Tumiotto, Camille; Riviere, Lionel; Bellecave, Pantxika; Recordon-Pinson, Patricia; Vilain-Parce, Alice; Guidicelli, Gwenda-Line; Fleury, Hervé

2017-01-01

One of the strategies for curing viral HIV-1 is a therapeutic vaccine involving the stimulation of cytotoxic CD8-positive T cells (CTL) that are Human Leucocyte Antigen (HLA)-restricted. The lack of efficiency of previous vaccination strategies may have been due to the immunogenic peptides used, which could be different from a patient's virus epitopes and lead to a poor CTL response. To counteract this lack of specificity, conserved epitopes must be targeted. One alternative is to gather as many data as possible from a large number of patients on their HIV-1 proviral archived epitope variants, taking into account their genetic background to select the best presented CTL epitopes. In order to process big data generated by Next-Generation Sequencing (NGS) of the DNA of HIV-infected patients, we have developed a software package called TutuGenetics. This tool combines an alignment derived either from Sanger or NGS files, HLA typing, target gene and a CTL epitope list as input files. It allows automatic translation after correction of the alignment obtained between the HxB2 reference and the reads, followed by automatic calculation of the MHC IC50 value for each epitope variant and the HLA allele of the patient by using NetMHCpan 3.0, resulting in a csv file as output result. We validated this new tool by comparing Sanger and NGS (454, Roche) sequences obtained from the proviral DNA of patients at success of ART included in the Provir Latitude 45 study and showed a 90% correlation between the quantitative results of NGS and Sanger. This automated analysis combined with complementary samples should yield more data regarding the archived CTL epitopes according to the patients' HLA alleles and will be useful for screening epitopes that in theory are presented efficiently to the HLA groove, thus constituting promising immunogenic peptides for a therapeutic vaccine.

Workflow and web application for annotating NCBI BioProject transcriptome data.

PubMed

Vera Alvarez, Roberto; Medeiros Vidal, Newton; Garzón-Martínez, Gina A; Barrero, Luz S; Landsman, David; Mariño-Ramírez, Leonardo

2017-01-01

The volume of transcriptome data is growing exponentially due to rapid improvement of experimental technologies. In response, large central resources such as those of the National Center for Biotechnology Information (NCBI) are continually adapting their computational infrastructure to accommodate this large influx of data. New and specialized databases, such as Transcriptome Shotgun Assembly Sequence Database (TSA) and Sequence Read Archive (SRA), have been created to aid the development and expansion of centralized repositories. Although the central resource databases are under continual development, they do not include automatic pipelines to increase annotation of newly deposited data. Therefore, third-party applications are required to achieve that aim. Here, we present an automatic workflow and web application for the annotation of transcriptome data. The workflow creates secondary data such as sequencing reads and BLAST alignments, which are available through the web application. They are based on freely available bioinformatics tools and scripts developed in-house. The interactive web application provides a search engine and several browser utilities. Graphical views of transcript alignments are available through SeqViewer, an embedded tool developed by NCBI for viewing biological sequence data. The web application is tightly integrated with other NCBI web applications and tools to extend the functionality of data processing and interconnectivity. We present a case study for the species Physalis peruviana with data generated from BioProject ID 67621. URL: http://www.ncbi.nlm.nih.gov/projects/physalis/. Published by Oxford University Press 2017. This work is written by US Government employees and is in the public domain in the US.

11. Photocopy of circa 1948 photograph showing detail view of ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

11. Photocopy of circa 1948 photograph showing detail view of Waiting Room ceiling, courtesy of Reading Company Archives - Philadelphia & Reading Railroad, Terminal Station, 1115-1141 Market Street, Philadelphia, Philadelphia County, PA

Evaluation of Targeted Sequencing for Transcriptional Analysis of Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples

EPA Science Inventory

Next-generation sequencing provides unprecedented access to genomic information in archival FFPE tissue samples. However, costs and technical challenges related to RNA isolation and enrichment limit use of whole-genome RNA-sequencing for large-scale studies of FFPE specimens. Rec...

Beams of particles and papers: How digital preprint archives shape authorship and credit.

PubMed

Delfanti, Alessandro

2016-08-01

In high energy physics, scholarly papers circulate primarily through online preprint archives based on a centralized repository, arXiv, that physicists simply refer to as 'the archive'. The archive is not just a tool for preservation and memory but also a space of flows where written objects are detected and their authors made available for scrutiny. In this article, I analyze the reading and publishing practices of two subsets of high energy physicists: theorists and experimentalists. In order to be recognized as legitimate and productive members of their community, they need to abide by the temporalities and authorial practices structured by the archive. Theorists live in a state of accelerated time that shapes their reading and publishing practices around precise cycles. Experimentalists turn to tactics that allow them to circumvent the slowed-down time and invisibility they experience as members of large collaborations. As digital platforms for the exchange of scholarly articles emerge in other fields, high energy physics could help shed light on general transformations of contemporary scholarly communication systems.

Rail-dbGaP: analyzing dbGaP-protected data in the cloud with Amazon Elastic MapReduce.

PubMed

Nellore, Abhinav; Wilks, Christopher; Hansen, Kasper D; Leek, Jeffrey T; Langmead, Ben

2016-08-15

Public archives contain thousands of trillions of bases of valuable sequencing data. More than 40% of the Sequence Read Archive is human data protected by provisions such as dbGaP. To analyse dbGaP-protected data, researchers must typically work with IT administrators and signing officials to ensure all levels of security are implemented at their institution. This is a major obstacle, impeding reproducibility and reducing the utility of archived data. We present a protocol and software tool for analyzing protected data in a commercial cloud. The protocol, Rail-dbGaP, is applicable to any tool running on Amazon Web Services Elastic MapReduce. The tool, Rail-RNA v0.2, is a spliced aligner for RNA-seq data, which we demonstrate by running on 9662 samples from the dbGaP-protected GTEx consortium dataset. The Rail-dbGaP protocol makes explicit for the first time the steps an investigator must take to develop Elastic MapReduce pipelines that analyse dbGaP-protected data in a manner compliant with NIH guidelines. Rail-RNA automates implementation of the protocol, making it easy for typical biomedical investigators to study protected RNA-seq data, regardless of their local IT resources or expertise. Rail-RNA is available from http://rail.bio Technical details on the Rail-dbGaP protocol as well as an implementation walkthrough are available at https://github.com/nellore/rail-dbgap Detailed instructions on running Rail-RNA on dbGaP-protected data using Amazon Web Services are available at http://docs.rail.bio/dbgap/ : anellore@gmail.com or langmea@cs.jhu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Quality scores for 32,000 genomes

DOE PAGES

Land, Miriam L.; Hyatt, Doug; Jun, Se-Ran; ...

2014-12-08

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). In this study, we have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences. Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes hadmore » quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes. The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Finally and unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.« less

Quality scores for 32,000 genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Land, Miriam L.; Hyatt, Doug; Jun, Se-Ran

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). In this study, we have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences. Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes hadmore » quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes. The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Finally and unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.« less

Sequence verification of synthetic DNA by assembly of sequencing reads

PubMed Central

Wilson, Mandy L.; Cai, Yizhi; Hanlon, Regina; Taylor, Samantha; Chevreux, Bastien; Setubal, João C.; Tyler, Brett M.; Peccoud, Jean

2013-01-01

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org. PMID:23042248

Light-weight reference-based compression of FASTQ data.

PubMed

Zhang, Yongpeng; Li, Linsen; Yang, Yanli; Yang, Xiao; He, Shan; Zhu, Zexuan

2015-06-09

The exponential growth of next generation sequencing (NGS) data has posed big challenges to data storage, management and archive. Data compression is one of the effective solutions, where reference-based compression strategies can typically achieve superior compression ratios compared to the ones not relying on any reference. This paper presents a lossless light-weight reference-based compression algorithm namely LW-FQZip to compress FASTQ data. The three components of any given input, i.e., metadata, short reads and quality score strings, are first parsed into three data streams in which the redundancy information are identified and eliminated independently. Particularly, well-designed incremental and run-length-limited encoding schemes are utilized to compress the metadata and quality score streams, respectively. To handle the short reads, LW-FQZip uses a novel light-weight mapping model to fast map them against external reference sequence(s) and produce concise alignment results for storage. The three processed data streams are then packed together with some general purpose compression algorithms like LZMA. LW-FQZip was evaluated on eight real-world NGS data sets and achieved compression ratios in the range of 0.111-0.201. This is comparable or superior to other state-of-the-art lossless NGS data compression algorithms. LW-FQZip is a program that enables efficient lossless FASTQ data compression. It contributes to the state of art applications for NGS data storage and transmission. LW-FQZip is freely available online at: http://csse.szu.edu.cn/staff/zhuzx/LWFQZip.

SraTailor: graphical user interface software for processing and visualizing ChIP-seq data.

PubMed

Oki, Shinya; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Meno, Chikara

2014-12-01

Raw data from ChIP-seq (chromatin immunoprecipitation combined with massively parallel DNA sequencing) experiments are deposited in public databases as SRAs (Sequence Read Archives) that are publically available to all researchers. However, to graphically visualize ChIP-seq data of interest, the corresponding SRAs must be downloaded and converted into BigWig format, a process that involves complicated command-line processing. This task requires users to possess skill with script languages and sequence data processing, a requirement that prevents a wide range of biologists from exploiting SRAs. To address these challenges, we developed SraTailor, a GUI (Graphical User Interface) software package that automatically converts an SRA into a BigWig-formatted file. Simplicity of use is one of the most notable features of SraTailor: entering an accession number of an SRA and clicking the mouse are the only steps required to obtain BigWig-formatted files and to graphically visualize the extents of reads at given loci. SraTailor is also able to make peak calls, generate files of other formats, process users' own data, and accept various command-line-like options. Therefore, this software makes ChIP-seq data fully exploitable by a wide range of biologists. SraTailor is freely available at http://www.devbio.med.kyushu-u.ac.jp/sra_tailor/, and runs on both Mac and Windows machines. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Illumina Synthetic Long Read Sequencing Allows Recovery of Missing Sequences even in the “Finished” C. elegans Genome

PubMed Central

Li, Runsheng; Hsieh, Chia-Ling; Young, Amanda; Zhang, Zhihong; Ren, Xiaoliang; Zhao, Zhongying

2015-01-01

Most next-generation sequencing platforms permit acquisition of high-throughput DNA sequences, but the relatively short read length limits their use in genome assembly or finishing. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking. We evaluate the promise and deficiency of the long reads in these aspects using isogenic C. elegans genome with no gap. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences prevents pre-assembly of long reads in the relevant genomic region. Second, the reads are able to reliably detect missing but not extra sequences in the C. elegans genome. Third, the reads of smaller size are more capable of recovering repetitive sequences than those of bigger size. Fourth, at least 40 Kbp missing genomic sequences are recovered in the C. elegans genome using the long reads. Finally, an N50 contig size of at least 86 Kbp can be achieved with 24×reads but with substantial mis-assembly errors, highlighting a need for novel assembly algorithm for the long reads. PMID:26039588

Summertime...and Reading Beckons.

ERIC Educational Resources Information Center

Bettmann, Otto

2000-01-01

Presents a collection of quotes by famous people about reading for enjoyment and personal development. The collection was assembled from a lifetime of fond association with books and reading by the rare-book librarian at the State Library in Berlin, who after Hitler's rise, relocated to the United States and founded the Bettmann Archive in New…

The Effects of Read 180 on Student Achievement

ERIC Educational Resources Information Center

Plony, Doreen A.

2013-01-01

The purpose of this ex post facto study was to analyze archive data to investigate the effects of Read 180, a computer-based supplemental reading intervention, on students' academic achievement for the academic school year 2011-2012. Further analyses examined if influences existed in variables such as grade level, gender, and ethnicity of the…

Database resources of the National Center for Biotechnology Information.

PubMed

2016-01-04

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank(®) nucleic acid sequence database and the PubMed database of citations and abstracts for published life science journals. Additional NCBI resources focus on literature (PubMed Central (PMC), Bookshelf and PubReader), health (ClinVar, dbGaP, dbMHC, the Genetic Testing Registry, HIV-1/Human Protein Interaction Database and MedGen), genomes (BioProject, Assembly, Genome, BioSample, dbSNP, dbVar, Epigenomics, the Map Viewer, Nucleotide, Probe, RefSeq, Sequence Read Archive, the Taxonomy Browser and the Trace Archive), genes (Gene, Gene Expression Omnibus (GEO), HomoloGene, PopSet and UniGene), proteins (Protein, the Conserved Domain Database (CDD), COBALT, Conserved Domain Architecture Retrieval Tool (CDART), the Molecular Modeling Database (MMDB) and Protein Clusters) and chemicals (Biosystems and the PubChem suite of small molecule databases). The Entrez system provides search and retrieval operations for most of these databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Database resources of the National Center for Biotechnology Information.

PubMed

2015-01-01

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank(®) nucleic acid sequence database and the PubMed database of citations and abstracts for published life science journals. Additional NCBI resources focus on literature (Bookshelf, PubMed Central (PMC) and PubReader); medical genetics (ClinVar, dbMHC, the Genetic Testing Registry, HIV-1/Human Protein Interaction Database and MedGen); genes and genomics (BioProject, BioSample, dbSNP, dbVar, Epigenomics, Gene, Gene Expression Omnibus (GEO), Genome, HomoloGene, the Map Viewer, Nucleotide, PopSet, Probe, RefSeq, Sequence Read Archive, the Taxonomy Browser, Trace Archive and UniGene); and proteins and chemicals (Biosystems, COBALT, the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), the Molecular Modeling Database (MMDB), Protein Clusters, Protein and the PubChem suite of small molecule databases). The Entrez system provides search and retrieval operations for many of these databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of these resources can be accessed through the NCBI home page at http://www.ncbi.nlm.nih.gov. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Dose-Response Analysis of RNA-Seq Profiles in Archival ...

EPA Pesticide Factsheets

Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from two archival studies in mice, one 20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively. Total RNA was ribo-depleted and sequenced using the Illumina HiSeq platform. In the recently archived study, FFPE samples had 35% lower total counts compared to FROZ samples but high concordance in fold-change values of differentially expressed genes (DEGs) (r2 = 0.99), highly enriched pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (2% difference vs FROZ). In contrast, older FFPE samples had markedly lower total counts (3% of FROZ) and poor concordance in global DEGs and pathways. However, counts from FFPE and FROZ samples still positively correlated (r2 = 0.84 across all transcripts) and showed comparable dose responses for more highly expressed target genes. These findings highlight potential applications and issues in using RNA-sequencing data from FFPE samples. Recently archived FFPE samples were highly similar to FROZ samples in sequencing q

10. Photocopy of circa 1948 photograph showing a view of ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

10. Photocopy of circa 1948 photograph showing a view of the Waiting Room Access of Train Shed, courtesy of Reading Company Archives - Philadelphia & Reading Railroad, Terminal Station, 1115-1141 Market Street, Philadelphia, Philadelphia County, PA

gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes.

PubMed

Nakagawa, So; Takahashi, Mahoko Ueda

2016-01-01

In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs), which are derived from ancient viral infections of germ cells. Although most EVEs have been inactivated, some open reading frames (ORFs) of EVEs obtained functions in the hosts. However, EVE ORFs usually remain unannotated in the genomes, and no databases are available for EVE ORFs. To investigate the function and evolution of EVEs in mammalian genomes, we developed EVE ORF databases for 20 genomes of 19 mammalian species. A total of 736,771 non-overlapping EVE ORFs were identified and archived in a database named gEVE (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs for all 20 genomes. In analyzing RNA-seq data with the gEVE database, we successfully identified the expressed EVE genes, suggesting that the gEVE database facilitates studies of the genomic analyses of various mammalian species.Database URL: http://geve.med.u-tokai.ac.jp. © The Author(s) 2016. Published by Oxford University Press.

A fully decompressed synthetic bacteriophage øX174 genome assembled and archived in yeast.

PubMed

Jaschke, Paul R; Lieberman, Erica K; Rodriguez, Jon; Sierra, Adrian; Endy, Drew

2012-12-20

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage. Copyright © 2012 Elsevier Inc. All rights reserved.

gEVE: a genome-based endogenous viral element database provides comprehensive viral protein-coding sequences in mammalian genomes

PubMed Central

Nakagawa, So; Takahashi, Mahoko Ueda

2016-01-01

In mammals, approximately 10% of genome sequences correspond to endogenous viral elements (EVEs), which are derived from ancient viral infections of germ cells. Although most EVEs have been inactivated, some open reading frames (ORFs) of EVEs obtained functions in the hosts. However, EVE ORFs usually remain unannotated in the genomes, and no databases are available for EVE ORFs. To investigate the function and evolution of EVEs in mammalian genomes, we developed EVE ORF databases for 20 genomes of 19 mammalian species. A total of 736,771 non-overlapping EVE ORFs were identified and archived in a database named gEVE (http://geve.med.u-tokai.ac.jp). The gEVE database provides nucleotide and amino acid sequences, genomic loci and functional annotations of EVE ORFs for all 20 genomes. In analyzing RNA-seq data with the gEVE database, we successfully identified the expressed EVE genes, suggesting that the gEVE database facilitates studies of the genomic analyses of various mammalian species. Database URL: http://geve.med.u-tokai.ac.jp PMID:27242033

Books and Pets: Our Friends for Life! Arizona Reading Program Manual.

ERIC Educational Resources Information Center

Arizona State Dept. of Library, Archives and Public Records, Phoenix.

This reading program manual delineates the "Books and Pets" program, a project of Arizona Reads, which is a collaboration between the Arizona Humanities Council and the Arizona State Library, Archives, and Public Records. A CD-ROM version of the program accompanies the manual. The manual is divided into the following parts: Introduction;…

Increasing Oral Reading Fluency of below Grade-Level Elementary Students through Parent Involvement

ERIC Educational Resources Information Center

Royal, Louise I.

2012-01-01

An increasing number of elementary students in a rural school were promoted to a higher grade without having grade-level reading fluency skills, thereby becoming at risk of not reaching or maintaining their academic grade level reading skills. The purpose of this ex post facto quantitative study involving archival data analysis was to investigate…

An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.

PubMed

Reading, Benjamin J; Chapman, Robert W; Schaff, Jennifer E; Scholl, Elizabeth H; Opperman, Charles H; Sullivan, Craig V

2012-02-21

The striped bass and its relatives (genus Morone) are important fisheries and aquaculture species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America. To open avenues of gene expression research on reproduction and breeding of striped bass, we generated a collection of expressed sequence tags (ESTs) from a complementary DNA (cDNA) library representative of their ovarian transcriptome. Sequences of a total of 230,151 ESTs (51,259,448 bp) were acquired by Roche 454 pyrosequencing of cDNA pooled from ovarian tissues obtained at all stages of oocyte growth, at ovulation (eggs), and during preovulatory atresia. Quality filtering of ESTs allowed assembly of 11,208 high-quality contigs ≥ 100 bp, including 2,984 contigs 500 bp or longer (average length 895 bp). Blastx comparisons revealed 5,482 gene orthologues (E-value < 10-3), of which 4,120 (36.7% of total contigs) were annotated with Gene Ontology terms (E-value < 10-6). There were 5,726 remaining unknown unique sequences (51.1% of total contigs). All of the high-quality EST sequences are available in the National Center for Biotechnology Information (NCBI) Short Read Archive (GenBank: SRX007394). Informative contigs were considered to be abundant if they were assembled from groups of ESTs comprising ≥ 0.15% of the total short read sequences (≥ 345 reads/contig). Approximately 52.5% of these abundant contigs were predicted to have predominant ovary expression through digital differential display in silico comparisons to zebrafish (Danio rerio) UniGene orthologues. Over 1,300 Gene Ontology terms from Biological Process classes of Reproduction, Reproductive process, and Developmental process were assigned to this collection of annotated contigs. This first large reference sequence database available for the ecologically and economically important temperate basses (genus Morone) provides a foundation for gene expression studies in these species. The predicted predominance of ovary gene expression and assignment of directly relevant Gene Ontology classes suggests a powerful utility of this dataset for analysis of ovarian gene expression related to fundamental questions of oogenesis. Additionally, a high definition Agilent 60-mer oligo ovary 'UniClone' microarray with 8 × 15,000 probe format has been designed based on this striped bass transcriptome (eArray Group: Striper Group, Design ID: 029004).

ALI--A Digital Archive of DAISY Books

ERIC Educational Resources Information Center

Forsberg, Asa

2007-01-01

ALI is a project to develop an archive for talking books produced by the Swedish universities. The universities produce talking books from the mandatory literature for students with reading disabilities, including mostly journal articles, book chapters and texts written by teachers. The project group consists of librarians and co-ordinators for…

Facilities Requirements for Archives and Special Collections Department.

ERIC Educational Resources Information Center

Brown, Charlotte B.

The program of the Archives and Special Collections Department at Franklin and Marshall College requires the following function areas to be located in the Shadek-Fackenthal Library: (1) Reading Room; (2) Conservation Laboratory; (3) Isolation Room; (4) storage for permanent collection; (5) storage for high security materials; (6) Processing Room;…

AmpliVar: mutation detection in high-throughput sequence from amplicon-based libraries.

PubMed

Hsu, Arthur L; Kondrashova, Olga; Lunke, Sebastian; Love, Clare J; Meldrum, Cliff; Marquis-Nicholson, Renate; Corboy, Greg; Pham, Kym; Wakefield, Matthew; Waring, Paul M; Taylor, Graham R

2015-04-01

Conventional means of identifying variants in high-throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key-value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low-abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon-based high-throughput sequencing data. The method can be extended to enable alignment-free confirmation of variants seen in hybridization capture target-enrichment data. © 2015 WILEY PERIODICALS, INC.

ISRNA: an integrative online toolkit for short reads from high-throughput sequencing data.

PubMed

Luo, Guan-Zheng; Yang, Wei; Ma, Ying-Ke; Wang, Xiu-Jie

2014-02-01

Integrative Short Reads NAvigator (ISRNA) is an online toolkit for analyzing high-throughput small RNA sequencing data. Besides the high-speed genome mapping function, ISRNA provides statistics for genomic location, length distribution and nucleotide composition bias analysis of sequence reads. Number of reads mapped to known microRNAs and other classes of short non-coding RNAs, coverage of short reads on genes, expression abundance of sequence reads as well as some other analysis functions are also supported. The versatile search functions enable users to select sequence reads according to their sub-sequences, expression abundance, genomic location, relationship to genes, etc. A specialized genome browser is integrated to visualize the genomic distribution of short reads. ISRNA also supports management and comparison among multiple datasets. ISRNA is implemented in Java/C++/Perl/MySQL and can be freely accessed at http://omicslab.genetics.ac.cn/ISRNA/.

Haplotype estimation using sequencing reads.

PubMed

Delaneau, Olivier; Howie, Bryan; Cox, Anthony J; Zagury, Jean-François; Marchini, Jonathan

2013-10-03

High-throughput sequencing technologies produce short sequence reads that can contain phase information if they span two or more heterozygote genotypes. This information is not routinely used by current methods that infer haplotypes from genotype data. We have extended the SHAPEIT2 method to use phase-informative sequencing reads to improve phasing accuracy. Our model incorporates the read information in a probabilistic model through base quality scores within each read. The method is primarily designed for high-coverage sequence data or data sets that already have genotypes called. One important application is phasing of single samples sequenced at high coverage for use in medical sequencing and studies of rare diseases. Our method can also use existing panels of reference haplotypes. We tested the method by using a mother-father-child trio sequenced at high-coverage by Illumina together with the low-coverage sequence data from the 1000 Genomes Project (1000GP). We found that use of phase-informative reads increases the mean distance between switch errors by 22% from 274.4 kb to 328.6 kb. We also used male chromosome X haplotypes from the 1000GP samples to simulate sequencing reads with varying insert size, read length, and base error rate. When using short 100 bp paired-end reads, we found that using mixtures of insert sizes produced the best results. When using longer reads with high error rates (5-20 kb read with 4%-15% error per base), phasing performance was substantially improved. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Read clouds uncover variation in complex regions of the human genome

PubMed Central

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E.; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-01-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. PMID:26286554

A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb

PubMed Central

Meng, Yijun; Yu, Dongliang; Xue, Jie; Lu, Jiangjie; Feng, Shangguo; Shen, Chenjia; Wang, Huizhong

2016-01-01

Dendrobium officinale is an important traditional Chinese herb. Here, we did a transcriptome-wide, organ-specific study on this valuable plant by combining RNA, small RNA (sRNA) and degradome sequencing. RNA sequencing of four organs (flower, root, leaf and stem) of Dendrobium officinale enabled us to obtain 536,558 assembled transcripts, from which 2,645, 256, 42 and 54 were identified to be highly expressed in the four organs respectively. Based on sRNA sequencing, 2,038, 2, 21 and 24 sRNAs were identified to be specifically accumulated in the four organs respectively. A total of 1,047 mature microRNA (miRNA) candidates were detected. Based on secondary structure predictions and sequencing, tens of potential miRNA precursors were identified from the assembled transcripts. Interestingly, phase-distributed sRNAs with degradome-based processing evidences were discovered on the long-stem structures of two precursors. Target identification was performed for the 1,047 miRNA candidates, resulting in the discovery of 1,257 miRNA--target pairs. Finally, some biological meaningful subnetworks involving hormone signaling, development, secondary metabolism and Argonaute 1-related regulation were established. All of the sequencing data sets are available at NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/). Summarily, our study provides a valuable resource for the in-depth molecular and functional studies on this important Chinese orchid herb. PMID:26732614

Use of Schema on Read in Earth Science Data Archives

NASA Astrophysics Data System (ADS)

Petrenko, M.; Hegde, M.; Smit, C.; Pilone, P.; Pham, L.

2017-12-01

Traditionally, NASA Earth Science data archives have file-based storage using proprietary data file formats, such as HDF and HDF-EOS, which are optimized to support fast and efficient storage of spaceborne and model data as they are generated. The use of file-based storage essentially imposes an indexing strategy based on data dimensions. In most cases, NASA Earth Science data uses time as the primary index, leading to poor performance in accessing data in spatial dimensions. For example, producing a time series for a single spatial grid cell involves accessing a large number of data files. With exponential growth in data volume due to the ever-increasing spatial and temporal resolution of the data, using file-based archives poses significant performance and cost barriers to data discovery and access. Storing and disseminating data in proprietary data formats imposes an additional access barrier for users outside the mainstream research community. At the NASA Goddard Earth Sciences Data Information Services Center (GES DISC), we have evaluated applying the "schema-on-read" principle to data access and distribution. We used Apache Parquet to store geospatial data, and have exposed data through Amazon Web Services (AWS) Athena, AWS Simple Storage Service (S3), and Apache Spark. Using the "schema-on-read" approach allows customization of indexing—spatial or temporal—to suit the data access pattern. The storage of data in open formats such as Apache Parquet has widespread support in popular programming languages. A wide range of solutions for handling big data lowers the access barrier for all users. This presentation will discuss formats used for data storage, frameworks with support for "schema-on-read" used for data access, and common use cases covering data usage patterns seen in a geospatial data archive.

Use of Schema on Read in Earth Science Data Archives

NASA Technical Reports Server (NTRS)

Hegde, Mahabaleshwara; Smit, Christine; Pilone, Paul; Petrenko, Maksym; Pham, Long

2017-01-01

Traditionally, NASA Earth Science data archives have file-based storage using proprietary data file formats, such as HDF and HDF-EOS, which are optimized to support fast and efficient storage of spaceborne and model data as they are generated. The use of file-based storage essentially imposes an indexing strategy based on data dimensions. In most cases, NASA Earth Science data uses time as the primary index, leading to poor performance in accessing data in spatial dimensions. For example, producing a time series for a single spatial grid cell involves accessing a large number of data files. With exponential growth in data volume due to the ever-increasing spatial and temporal resolution of the data, using file-based archives poses significant performance and cost barriers to data discovery and access. Storing and disseminating data in proprietary data formats imposes an additional access barrier for users outside the mainstream research community. At the NASA Goddard Earth Sciences Data Information Services Center (GES DISC), we have evaluated applying the schema-on-read principle to data access and distribution. We used Apache Parquet to store geospatial data, and have exposed data through Amazon Web Services (AWS) Athena, AWS Simple Storage Service (S3), and Apache Spark. Using the schema-on-read approach allows customization of indexing spatially or temporally to suit the data access pattern. The storage of data in open formats such as Apache Parquet has widespread support in popular programming languages. A wide range of solutions for handling big data lowers the access barrier for all users. This presentation will discuss formats used for data storage, frameworks with This presentation will discuss formats used for data storage, frameworks with support for schema-on-read used for data access, and common use cases covering data usage patterns seen in a geospatial data archive.

Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue

PubMed Central

Carrick, Danielle Mercatante; Mehaffey, Michele G.; Sachs, Michael C.; Altekruse, Sean; Camalier, Corinne; Chuaqui, Rodrigo; Cozen, Wendy; Das, Biswajit; Hernandez, Brenda Y.; Lih, Chih-Jian; Lynch, Charles F.; Makhlouf, Hala; McGregor, Paul; McShane, Lisa M.; Phillips Rohan, JoyAnn; Walsh, William D.; Williams, Paul M.; Gillanders, Elizabeth M.; Mechanic, Leah E.; Schully, Sheri D.

2015-01-01

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

JVM: Java Visual Mapping tool for next generation sequencing read.

PubMed

Yang, Ye; Liu, Juan

2015-01-01

We developed a program JVM (Java Visual Mapping) for mapping next generation sequencing read to reference sequence. The program is implemented in Java and is designed to deal with millions of short read generated by sequence alignment using the Illumina sequencing technology. It employs seed index strategy and octal encoding operations for sequence alignments. JVM is useful for DNA-Seq, RNA-Seq when dealing with single-end resequencing. JVM is a desktop application, which supports reads capacity from 1 MB to 10 GB.

Home Economics Archive: Research, Tradition and History (HEARTH)

Science.gov Websites

, Tradition and History HEARTH is a core electronic collection of books and journals in Home Economics and Intimate History of American Girls. Additional information, images and readings on the history of Home Archive: Research, Tradition and History (HEARTH). Ithaca, NY: Albert R. Mann Library, Cornell University

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS

PubMed Central

Jiao, Xiaoli; Zheng, Xin; Ma, Liang; Kutty, Geetha; Gogineni, Emile; Sun, Qiang; Sherman, Brad T.; Hu, Xiaojun; Jones, Kristine; Raley, Castle; Tran, Bao; Munroe, David J.; Stephens, Robert; Liang, Dun; Imamichi, Tomozumi; Kovacs, Joseph A.; Lempicki, Richard A.; Huang, Da Wei

2013-01-01

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results. PMID:24179701

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

PubMed

Jiao, Xiaoli; Zheng, Xin; Ma, Liang; Kutty, Geetha; Gogineni, Emile; Sun, Qiang; Sherman, Brad T; Hu, Xiaojun; Jones, Kristine; Raley, Castle; Tran, Bao; Munroe, David J; Stephens, Robert; Liang, Dun; Imamichi, Tomozumi; Kovacs, Joseph A; Lempicki, Richard A; Huang, Da Wei

2013-07-31

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.

Read clouds uncover variation in complex regions of the human genome.

PubMed

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-10-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. © 2015 Bishara et al.; Published by Cold Spring Harbor Laboratory Press.

Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples.

PubMed

Kresse, Stine H; Namløs, Heidi M; Lorenz, Susanne; Berner, Jeanne-Marie; Myklebost, Ola; Bjerkehagen, Bodil; Meza-Zepeda, Leonardo A

2018-01-01

Nucleic acid material of adequate quality is crucial for successful high-throughput sequencing (HTS) analysis. DNA and RNA isolated from archival FFPE material are frequently degraded and not readily amplifiable due to chemical damage introduced during fixation. To identify optimal nucleic acid extraction kits, DNA and RNA quantity, quality and performance in HTS applications were evaluated. DNA and RNA were isolated from five sarcoma archival FFPE blocks, using eight extraction protocols from seven kits from three different commercial vendors. For DNA extraction, the truXTRAC FFPE DNA kit from Covaris gave higher yields and better amplifiable DNA, but all protocols gave comparable HTS library yields using Agilent SureSelect XT and performed well in downstream variant calling. For RNA extraction, all protocols gave comparable yields and amplifiable RNA. However, for fusion gene detection using the Archer FusionPlex Sarcoma Assay, the truXTRAC FFPE RNA kit from Covaris and Agencourt FormaPure kit from Beckman Coulter showed the highest percentage of unique read-pairs, providing higher complexity of HTS data and more frequent detection of recurrent fusion genes. truXTRAC simultaneous DNA and RNA extraction gave similar outputs as individual protocols. These findings show that although successful HTS libraries could be generated in most cases, the different protocols gave variable quantity and quality for FFPE nucleic acid extraction. Selecting the optimal procedure is highly valuable and may generate results in borderline quality specimens.

Evaluating Quality of Aged Archival Formalin-Fixed Paraffin-Embedded Samples for RNA-Sequencing

EPA Science Inventory

Archival formalin-fixed paraffin-embedded (FFPE) samples offer a vast, untapped source of genomic data for biomarker discovery. However, the quality of FFPE samples is often highly variable, and conventional methods to assess RNA quality for RNA-sequencing (RNA-seq) are not infor...

Differential gene expression in the siphonophore Nanomia bijuga (Cnidaria) assessed with multiple next-generation sequencing workflows.

PubMed

Siebert, Stefan; Robinson, Mark D; Tintori, Sophia C; Goetz, Freya; Helm, Rebecca R; Smith, Stephen A; Shaner, Nathan; Haddock, Steven H D; Dunn, Casey W

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

PubMed Central

Siebert, Stefan; Robinson, Mark D.; Tintori, Sophia C.; Goetz, Freya; Helm, Rebecca R.; Smith, Stephen A.; Shaner, Nathan; Haddock, Steven H. D.; Dunn, Casey W.

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing. PMID:21829563

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

Developing English and Spanish Literacy in a One-Way Spanish Immersion Program

ERIC Educational Resources Information Center

Hollingsworth, Lindsay Kay

2013-01-01

This quantitative, causal-comparative study examined the possible cause and effect relationship between educational programming, specifically one-way Spanish immersion and traditional English-only, and native English-speaking fifth graders' vocabulary and reading comprehension. Archival data was used to examine students' reading achievement as…

E-Book versus Printed Materials: Preferences of University Students

ERIC Educational Resources Information Center

Cumaoglu, Gonca; Sacici, Esra; Torun, Kerem

2013-01-01

Reading habits, accessing resources, and material preferences change rapidly in a digital world. University students, as digital natives, are accessing countless resources, from lecture notes to research papers electronically. The change of reading habits with a great scale has led to differentiation on accessibility of resources, archiving them…

The Final Barrier: Security Consideration in Restricted Access Reading Rooms.

ERIC Educational Resources Information Center

Strassberg, Richard

1997-01-01

Examines an effective response to library or archive theft and vandalism of valuable materials: the restricted access reading room. Discusses the need for an alert staff, user identification, restriction of carry-in items, electronic surveillance, record keeping, limits to quantities of collection materials, exiting procedure, photocopying, theft…

Stories We've Told: 50 Years of CRLA Archives and Histories

ERIC Educational Resources Information Center

O'Donnell Lussier, Kristie; Harper Shetron, Tamara

2018-01-01

The purpose of this historical overview of the College Reading and Learning Association (CRLA) is to examine the CRLA archives to determine phases in the organization's narrative development, to uncover trends, and to ascertain overarching principles in the dialog between the written record and living memory. The two-phase research study began…

Library and Archival Security: Policies and Procedures To Protect Holdings from Theft and Damage.

ERIC Educational Resources Information Center

Trinkaus-Randall, Gregor

1998-01-01

Firm policies and procedures that address the environment, patron/staff behavior, general attitude, and care and handling of materials need to be at the core of the library/archival security program. Discussion includes evaluating a repository's security needs, collections security, security in non-public areas, security in the reading room,…

An efficient and scalable graph modeling approach for capturing information at different levels in next generation sequencing reads

PubMed Central

2013-01-01

Background Next generation sequencing technologies have greatly advanced many research areas of the biomedical sciences through their capability to generate massive amounts of genetic information at unprecedented rates. The advent of next generation sequencing has led to the development of numerous computational tools to analyze and assemble the millions to billions of short sequencing reads produced by these technologies. While these tools filled an important gap, current approaches for storing, processing, and analyzing short read datasets generally have remained simple and lack the complexity needed to efficiently model the produced reads and assemble them correctly. Results Previously, we presented an overlap graph coarsening scheme for modeling read overlap relationships on multiple levels. Most current read assembly and analysis approaches use a single graph or set of clusters to represent the relationships among a read dataset. Instead, we use a series of graphs to represent the reads and their overlap relationships across a spectrum of information granularity. At each information level our algorithm is capable of generating clusters of reads from the reduced graph, forming an integrated graph modeling and clustering approach for read analysis and assembly. Previously we applied our algorithm to simulated and real 454 datasets to assess its ability to efficiently model and cluster next generation sequencing data. In this paper we extend our algorithm to large simulated and real Illumina datasets to demonstrate that our algorithm is practical for both sequencing technologies. Conclusions Our overlap graph theoretic algorithm is able to model next generation sequencing reads at various levels of granularity through the process of graph coarsening. Additionally, our model allows for efficient representation of the read overlap relationships, is scalable for large datasets, and is practical for both Illumina and 454 sequencing technologies. PMID:24564333

De Novo Transcriptome Assembly and Characterization of the Synthesis Genes of Bioactive Constituents in Abelmoschus esculentus (L.) Moench

PubMed Central

Zhang, Chenghao; Dong, Wenqi; Gen, Wei; Xu, Baoyu; Shen, Chenjia

2018-01-01

Abelmoschus esculentus (okra or lady’s fingers) is a vegetable with high nutritional value, as well as having certain medicinal effects. It is widely used as food, in the food industry, and in herbal medicinal products, but also as an ornamental, in animal feed, and in other commercial sectors. Okra is rich in bioactive compounds, such as flavonoids, polysaccharides, polyphenols, caffeine, and pectin. In the present study, the concentrations of total flavonoids and polysaccharides in five organs of okra were determined and compared. Transcriptome sequencing was used to explore the biosynthesis pathways associated with the active constituents in okra. Transcriptome sequencing of five organs (roots, stem, leaves, flowers, and fruits) of okra enabled us to obtain 293,971 unigenes, of which 232,490 were annotated. Unigenes related to the enzymes involved in the flavonoid biosynthetic pathway or in fructose and mannose metabolism were identified, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. All of the transcriptional datasets were uploaded to Sequence Read Archive (SRA). In summary, our comprehensive analysis provides important information at the molecular level about the flavonoid and polysaccharide biosynthesis pathways in okra. PMID:29495525

Unlocking Short Read Sequencing for Metagenomics

DOE PAGES

Rodrigue, Sébastien; Materna, Arne C.; Timberlake, Sonia C.; ...

2010-07-28

We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.

Development of Genomic Simple Sequence Repeats (SSR) by Enrichment Libraries in Date Palm.

PubMed

Al-Faifi, Sulieman A; Migdadi, Hussein M; Algamdi, Salem S; Khan, Mohammad Altaf; Al-Obeed, Rashid S; Ammar, Megahed H; Jakse, Jerenj

2017-01-01

Development of highly informative markers such as simple sequence repeats (SSR) for cultivar identification and germplasm characterization and management is essential for date palms genetic studies. The present study documents the development of SSR markers and assesses genetic relationships of commonly grown date palm (Phoenix dactylifera L.) cultivars in different geographical regions of Saudi Arabia. A total of 93 novel simple sequence repeat (SSR) markers were screened for their ability to detect polymorphism in date palm. Around 71% of genomic SSRs are dinucleotide, 25% trinucleotide, 3% tetranucleotide, and 1% pentanucleotide motives and show 100% polymorphism. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis illustrates that cultivars trend to group according to their class of maturity, region of cultivation, and fruit color. Analysis of molecular variations (AMOVA) reveals genetic variation among and within cultivars of 27% and 73%, respectively, according to the geographical distribution of the cultivars. Developed microsatellite markers are of additional value to date palm characterization, tools which can be used by researchers in population genetics, cultivar identification, as well as genetic resource exploration and management. The cultivars tested exhibited a significant amount of genetic diversity and could be suitable for successful breeding programs. Genomic sequences generated from this study are available at the National Center for Biotechnology Information (NCBI), Sequence Read Archive (Accession numbers. LIBGSS_039019).

Long-read sequencing and de novo assembly of a Chinese genome

USDA-ARS?s Scientific Manuscript database

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arr...

FMLRC: Hybrid long read error correction using an FM-index.

PubMed

Wang, Jeremy R; Holt, James; McMillan, Leonard; Jones, Corbin D

2018-02-09

Long read sequencing is changing the landscape of genomic research, especially de novo assembly. Despite the high error rate inherent to long read technologies, increased read lengths dramatically improve the continuity and accuracy of genome assemblies. However, the cost and throughput of these technologies limits their application to complex genomes. One solution is to decrease the cost and time to assemble novel genomes by leveraging "hybrid" assemblies that use long reads for scaffolding and short reads for accuracy. We describe a novel method leveraging a multi-string Burrows-Wheeler Transform with auxiliary FM-index to correct errors in long read sequences using a set of complementary short reads. We demonstrate that our method efficiently produces significantly more high quality corrected sequence than existing hybrid error-correction methods. We also show that our method produces more contiguous assemblies, in many cases, than existing state-of-the-art hybrid and long-read only de novo assembly methods. Our method accurately corrects long read sequence data using complementary short reads. We demonstrate higher total throughput of corrected long reads and a corresponding increase in contiguity of the resulting de novo assemblies. Improved throughput and computational efficiency than existing methods will help better economically utilize emerging long read sequencing technologies.

JANE: efficient mapping of prokaryotic ESTs and variable length sequence reads on related template genomes

PubMed Central

2009-01-01

Background ESTs or variable sequence reads can be available in prokaryotic studies well before a complete genome is known. Use cases include (i) transcriptome studies or (ii) single cell sequencing of bacteria. Without suitable software their further analysis and mapping would have to await finalization of the corresponding genome. Results The tool JANE rapidly maps ESTs or variable sequence reads in prokaryotic sequencing and transcriptome efforts to related template genomes. It provides an easy-to-use graphics interface for information retrieval and a toolkit for EST or nucleotide sequence function prediction. Furthermore, we developed for rapid mapping an enhanced sequence alignment algorithm which reassembles and evaluates high scoring pairs provided from the BLAST algorithm. Rapid assembly on and replacement of the template genome by sequence reads or mapped ESTs is achieved. This is illustrated (i) by data from Staphylococci as well as from a Blattabacteria sequencing effort, (ii) mapping single cell sequencing reads is shown for poribacteria to sister phylum representative Rhodopirellula Baltica SH1. The algorithm has been implemented in a web-server accessible at http://jane.bioapps.biozentrum.uni-wuerzburg.de. Conclusion Rapid prokaryotic EST mapping or mapping of sequence reads is achieved applying JANE even without knowing the cognate genome sequence. PMID:19943962

Project MICAS: a multivendor open-system incremental approach to implementing an integrated enterprise-wide PACS: works in progress

NASA Astrophysics Data System (ADS)

Smith, Edward M.; Wright, Jeffrey; Fontaine, Marc T.; Robinson, Arvin E.

1998-07-01

The Medical Information, Communication and Archive System (MICAS) is a multi-vendor incremental approach to PACS. MICAS is a multi-modality integrated image management system that incorporates the radiology information system (RIS) and radiology image database (RID) with future 'hooks' to other hospital databases. Even though this approach to PACS is more risky than a single-vendor turn-key approach, it offers significant advantages. The vendors involved in the initial phase of MICAS are IDX Corp., ImageLabs, Inc. and Digital Equipment Corp (DEC). The network architecture operates at 100 MBits per sec except between the modalities and the stackable intelligent switch which is used to segment MICAS by modality. Each modality segment contains the acquisition engine for the modality, a temporary archive and one or more diagnostic workstations. All archived studies are available at all workstations, but there is no permanent archive at this time. At present, the RIS vendor is responsible for study acquisition and workflow as well as maintenance of the temporary archive. Management of study acquisition, workflow and the permanent archive will become the responsibility of the archive vendor when the archive is installed in the second quarter of 1998. The modalities currently interfaced to MICAS are MRI, CT and a Howtek film digitizer with Nuclear Medicine and computed radiography (CR) to be added when the permanent archive is installed. There are six dual-monitor diagnostic workstations which use ImageLabs Shared Vision viewer software located in MRI, CT, Nuclear Medicine, musculoskeletal reading areas and two in Radiology's main reading area. One of the major lessons learned to date is that the permanent archive should have been part of the initial MICAS installation and the archive vendor should have been responsible for image acquisition rather than the RIS vendor. Currently an archive vendor is being selected who will be responsible for the management of the archive plus the HIS/RIS interface, image acquisition, modality work list manager and interfacing to the current DICOM viewer software. The next phase of MICAS will include interfacing ultrasound, locating servers outside of the Radiology LAN to support the distribution of images and reports to the clinical floors and physician offices both within and outside of the University of Rochester Medical Center (URMC) campus and the teaching archive.

A Test of the Relationship between Reading Ability & Standardized Biology Assessment Scores

ERIC Educational Resources Information Center

Allen, Denise A.

2014-01-01

Little empirical evidence suggested that independent reading abilities of students enrolled in biology predicted their performance on the Biology I Graduation End-of-Course Assessment (ECA). An archival study was conducted at one Indiana urban public high school in Indianapolis, Indiana, by examining existing educational assessment data to test…

The Digitization of Early English Books: A Database Comparison of Internet Archive and Early English Books Online

ERIC Educational Resources Information Center

Brightenburg, Cindy

2016-01-01

The use of digital books is diverse, ranging from casual reading to in-depth primary source research. Digitization of early English printed books in particular, has provided greater access to a previously limited resource for academic faculty and researchers. Internet Archive, a free, internet website and Early English Books Online, a subscription…

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

PubMed

Blanchard, Adam M; Jolley, Keith A; Maiden, Martin C J; Coffey, Tracey J; Maboni, Grazieli; Staley, Ceri E; Bollard, Nicola J; Warry, Andrew; Emes, Richard D; Davies, Peers L; Tötemeyer, Sabine

2018-01-01

Dichelobacter nodosus ( D. nodosus ) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly.

PubMed

Koren, Sergey; Phillippy, Adam M

2015-02-01

Like a jigsaw puzzle with large pieces, a genome sequenced with long reads is easier to assemble. However, recent sequencing technologies have favored lowering per-base cost at the expense of read length. This has dramatically reduced sequencing cost, but resulted in fragmented assemblies, which negatively affect downstream analyses and hinder the creation of finished (gapless, high-quality) genomes. In contrast, emerging long-read sequencing technologies can now produce reads tens of kilobases in length, enabling the automated finishing of microbial genomes for under $1000. This promises to improve the quality of reference databases and facilitate new studies of chromosomal structure and variation. We present an overview of these new technologies and the methods used to assemble long reads into complete genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

TagDust2: a generic method to extract reads from sequencing data.

PubMed

Lassmann, Timo

2015-01-28

Arguably the most basic step in the analysis of next generation sequencing data (NGS) involves the extraction of mappable reads from the raw reads produced by sequencing instruments. The presence of barcodes, adaptors and artifacts subject to sequencing errors makes this step non-trivial. Here I present TagDust2, a generic approach utilizing a library of hidden Markov models (HMM) to accurately extract reads from a wide array of possible read architectures. TagDust2 extracts more reads of higher quality compared to other approaches. Processing of multiplexed single, paired end and libraries containing unique molecular identifiers is fully supported. Two additional post processing steps are included to exclude known contaminants and filter out low complexity sequences. Finally, TagDust2 can automatically detect the library type of sequenced data from a predefined selection. Taken together TagDust2 is a feature rich, flexible and adaptive solution to go from raw to mappable NGS reads in a single step. The ability to recognize and record the contents of raw reads will help to automate and demystify the initial, and often poorly documented, steps in NGS data analysis pipelines. TagDust2 is freely available at: http://tagdust.sourceforge.net .

Anatomy of a hash-based long read sequence mapping algorithm for next generation DNA sequencing.

PubMed

Misra, Sanchit; Agrawal, Ankit; Liao, Wei-keng; Choudhary, Alok

2011-01-15

Recently, a number of programs have been proposed for mapping short reads to a reference genome. Many of them are heavily optimized for short-read mapping and hence are very efficient for shorter queries, but that makes them inefficient or not applicable for reads longer than 200 bp. However, many sequencers are already generating longer reads and more are expected to follow. For long read sequence mapping, there are limited options; BLAT, SSAHA2, FANGS and BWA-SW are among the popular ones. However, resequencing and personalized medicine need much faster software to map these long sequencing reads to a reference genome to identify SNPs or rare transcripts. We present AGILE (AliGnIng Long rEads), a hash table based high-throughput sequence mapping algorithm for longer 454 reads that uses diagonal multiple seed-match criteria, customized q-gram filtering and a dynamic incremental search approach among other heuristics to optimize every step of the mapping process. In our experiments, we observe that AGILE is more accurate than BLAT, and comparable to BWA-SW and SSAHA2. For practical error rates (< 5%) and read lengths (200-1000 bp), AGILE is significantly faster than BLAT, SSAHA2 and BWA-SW. Even for the other cases, AGILE is comparable to BWA-SW and several times faster than BLAT and SSAHA2. http://www.ece.northwestern.edu/~smi539/agile.html.

Atropos: specific, sensitive, and speedy trimming of sequencing reads.

PubMed

Didion, John P; Martin, Marcel; Collins, Francis S

2017-01-01

A key step in the transformation of raw sequencing reads into biological insights is the trimming of adapter sequences and low-quality bases. Read trimming has been shown to increase the quality and reliability while decreasing the computational requirements of downstream analyses. Many read trimming software tools are available; however, no tool simultaneously provides the accuracy, computational efficiency, and feature set required to handle the types and volumes of data generated in modern sequencing-based experiments. Here we introduce Atropos and show that it trims reads with high sensitivity and specificity while maintaining leading-edge speed. Compared to other state-of-the-art read trimming tools, Atropos achieves significant increases in trimming accuracy while remaining competitive in execution times. Furthermore, Atropos maintains high accuracy even when trimming data with elevated rates of sequencing errors. The accuracy, high performance, and broad feature set offered by Atropos makes it an appropriate choice for the pre-processing of Illumina, ABI SOLiD, and other current-generation short-read sequencing datasets. Atropos is open source and free software written in Python (3.3+) and available at https://github.com/jdidion/atropos.

Atropos: specific, sensitive, and speedy trimming of sequencing reads

PubMed Central

Collins, Francis S.

2017-01-01

A key step in the transformation of raw sequencing reads into biological insights is the trimming of adapter sequences and low-quality bases. Read trimming has been shown to increase the quality and reliability while decreasing the computational requirements of downstream analyses. Many read trimming software tools are available; however, no tool simultaneously provides the accuracy, computational efficiency, and feature set required to handle the types and volumes of data generated in modern sequencing-based experiments. Here we introduce Atropos and show that it trims reads with high sensitivity and specificity while maintaining leading-edge speed. Compared to other state-of-the-art read trimming tools, Atropos achieves significant increases in trimming accuracy while remaining competitive in execution times. Furthermore, Atropos maintains high accuracy even when trimming data with elevated rates of sequencing errors. The accuracy, high performance, and broad feature set offered by Atropos makes it an appropriate choice for the pre-processing of Illumina, ABI SOLiD, and other current-generation short-read sequencing datasets. Atropos is open source and free software written in Python (3.3+) and available at https://github.com/jdidion/atropos. PMID:28875074

A framework for organizing cancer-related variations from existing databases, publications and NGS data using a High-performance Integrated Virtual Environment (HIVE).

PubMed

Wu, Tsung-Jung; Shamsaddini, Amirhossein; Pan, Yang; Smith, Krista; Crichton, Daniel J; Simonyan, Vahan; Mazumder, Raja

2014-01-01

Years of sequence feature curation by UniProtKB/Swiss-Prot, PIR-PSD, NCBI-CDD, RefSeq and other database biocurators has led to a rich repository of information on functional sites of genes and proteins. This information along with variation-related annotation can be used to scan human short sequence reads from next-generation sequencing (NGS) pipelines for presence of non-synonymous single-nucleotide variations (nsSNVs) that affect functional sites. This and similar workflows are becoming more important because thousands of NGS data sets are being made available through projects such as The Cancer Genome Atlas (TCGA), and researchers want to evaluate their biomarkers in genomic data. BioMuta, an integrated sequence feature database, provides a framework for automated and manual curation and integration of cancer-related sequence features so that they can be used in NGS analysis pipelines. Sequence feature information in BioMuta is collected from the Catalogue of Somatic Mutations in Cancer (COSMIC), ClinVar, UniProtKB and through biocuration of information available from publications. Additionally, nsSNVs identified through automated analysis of NGS data from TCGA are also included in the database. Because of the petabytes of data and information present in NGS primary repositories, a platform HIVE (High-performance Integrated Virtual Environment) for storing, analyzing, computing and curating NGS data and associated metadata has been developed. Using HIVE, 31 979 nsSNVs were identified in TCGA-derived NGS data from breast cancer patients. All variations identified through this process are stored in a Curated Short Read archive, and the nsSNVs from the tumor samples are included in BioMuta. Currently, BioMuta has 26 cancer types with 13 896 small-scale and 308 986 large-scale study-derived variations. Integration of variation data allows identifications of novel or common nsSNVs that can be prioritized in validation studies. Database URL: BioMuta: http://hive.biochemistry.gwu.edu/tools/biomuta/index.php; CSR: http://hive.biochemistry.gwu.edu/dna.cgi?cmd=csr; HIVE: http://hive.biochemistry.gwu.edu.

A better sequence-read simulator program for metagenomics.

PubMed

Johnson, Stephen; Trost, Brett; Long, Jeffrey R; Pittet, Vanessa; Kusalik, Anthony

2014-01-01

There are many programs available for generating simulated whole-genome shotgun sequence reads. The data generated by many of these programs follow predefined models, which limits their use to the authors' original intentions. For example, many models assume that read lengths follow a uniform or normal distribution. Other programs generate models from actual sequencing data, but are limited to reads from single-genome studies. To our knowledge, there are no programs that allow a user to generate simulated data following non-parametric read-length distributions and quality profiles based on empirically-derived information from metagenomics sequencing data. We present BEAR (Better Emulation for Artificial Reads), a program that uses a machine-learning approach to generate reads with lengths and quality values that closely match empirically-derived distributions. BEAR can emulate reads from various sequencing platforms, including Illumina, 454, and Ion Torrent. BEAR requires minimal user input, as it automatically determines appropriate parameter settings from user-supplied data. BEAR also uses a unique method for deriving run-specific error rates, and extracts useful statistics from the metagenomic data itself, such as quality-error models. Many existing simulators are specific to a particular sequencing technology; however, BEAR is not restricted in this way. Because of its flexibility, BEAR is particularly useful for emulating the behaviour of technologies like Ion Torrent, for which no dedicated sequencing simulators are currently available. BEAR is also the first metagenomic sequencing simulator program that automates the process of generating abundances, which can be an arduous task. BEAR is useful for evaluating data processing tools in genomics. It has many advantages over existing comparable software, such as generating more realistic reads and being independent of sequencing technology, and has features particularly useful for metagenomics work.

Normal and compound poisson approximations for pattern occurrences in NGS reads.

PubMed

Zhai, Zhiyuan; Reinert, Gesine; Song, Kai; Waterman, Michael S; Luan, Yihui; Sun, Fengzhu

2012-06-01

Next generation sequencing (NGS) technologies are now widely used in many biological studies. In NGS, sequence reads are randomly sampled from the genome sequence of interest. Most computational approaches for NGS data first map the reads to the genome and then analyze the data based on the mapped reads. Since many organisms have unknown genome sequences and many reads cannot be uniquely mapped to the genomes even if the genome sequences are known, alternative analytical methods are needed for the study of NGS data. Here we suggest using word patterns to analyze NGS data. Word pattern counting (the study of the probabilistic distribution of the number of occurrences of word patterns in one or multiple long sequences) has played an important role in molecular sequence analysis. However, no studies are available on the distribution of the number of occurrences of word patterns in NGS reads. In this article, we build probabilistic models for the background sequence and the sampling process of the sequence reads from the genome. Based on the models, we provide normal and compound Poisson approximations for the number of occurrences of word patterns from the sequence reads, with bounds on the approximation error. The main challenge is to consider the randomness in generating the long background sequence, as well as in the sampling of the reads using NGS. We show the accuracy of these approximations under a variety of conditions for different patterns with various characteristics. Under realistic assumptions, the compound Poisson approximation seems to outperform the normal approximation in most situations. These approximate distributions can be used to evaluate the statistical significance of the occurrence of patterns from NGS data. The theory and the computational algorithm for calculating the approximate distributions are then used to analyze ChIP-Seq data using transcription factor GABP. Software is available online (www-rcf.usc.edu/∼fsun/Programs/NGS_motif_power/NGS_motif_power.html). In addition, Supplementary Material can be found online (www.liebertonline.com/cmb).

Mapping and phasing of structural variation in patient genomes using nanopore sequencing.

PubMed

Cretu Stancu, Mircea; van Roosmalen, Markus J; Renkens, Ivo; Nieboer, Marleen M; Middelkamp, Sjors; de Ligt, Joep; Pregno, Giulia; Giachino, Daniela; Mandrile, Giorgia; Espejo Valle-Inclan, Jose; Korzelius, Jerome; de Bruijn, Ewart; Cuppen, Edwin; Talkowski, Michael E; Marschall, Tobias; de Ridder, Jeroen; Kloosterman, Wigard P

2017-11-06

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

Hybrid error correction and de novo assembly of single-molecule sequencing reads

PubMed Central

Koren, Sergey; Schatz, Michael C.; Walenz, Brian P.; Martin, Jeffrey; Howard, Jason; Ganapathy, Ganeshkumar; Wang, Zhong; Rasko, David A.; McCombie, W. Richard; Jarvis, Erich D.; Phillippy, Adam M.

2012-01-01

Emerging single-molecule sequencing instruments can generate multi-kilobase sequences with the potential to dramatically improve genome and transcriptome assembly. However, the high error rate of single-molecule reads is challenging, and has limited their use to resequencing bacteria. To address this limitation, we introduce a novel correction algorithm and assembly strategy that utilizes shorter, high-identity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on Pacbio RS reads of phage, prokaryotic, and eukaryotic whole genomes, including the novel genome of the parrot Melopsittacus undulatus, as well as for RNA-seq reads of the corn (Zea mays) transcriptome. Our approach achieves over 99.9% read correction accuracy and produces substantially better assemblies than current sequencing strategies: in the best example, quintupling the median contig size relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly. PMID:22750884

De novo assembly of human genomes with massively parallel short read sequencing.

PubMed

Li, Ruiqiang; Zhu, Hongmei; Ruan, Jue; Qian, Wubin; Fang, Xiaodong; Shi, Zhongbin; Li, Yingrui; Li, Shengting; Shan, Gao; Kristiansen, Karsten; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

2010-02-01

Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.

Reads2Type: a web application for rapid microbial taxonomy identification.

PubMed

Saputra, Dhany; Rasmussen, Simon; Larsen, Mette V; Haddad, Nizar; Sperotto, Maria Maddalena; Aarestrup, Frank M; Lund, Ole; Sicheritz-Pontén, Thomas

2015-11-25

Identification of bacteria may be based on sequencing and molecular analysis of a specific locus such as 16S rRNA, or a set of loci such as in multilocus sequence typing. In the near future, healthcare institutions and routine diagnostic microbiology laboratories may need to sequence the entire genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete genomes. Using a dataset of 1003 whole genome sequenced bacteria from various sequencing platforms, Reads2Type was able to identify the species with 99.5 % accuracy and on the minutes time scale. In comparison with other tools, Reads2Type offers the advantage of not needing to transfer sequencing files, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html.

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

PubMed

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

Performing an Archive of Resistance: Challenging Normative Life Narratives through Literary Reading and Memoir Writing Research

ERIC Educational Resources Information Center

Robson, Claire; Sumara, Dennis; Luce-Kapler, Rebecca

2011-01-01

This research explores the ways in which normative structures organize experiences and representations of identities. It reports on two groups, one in which the members identified as rural and heterosexual and the other as urban and lesbian. Both participated in literary reading and response practices organized by a literary anthropological…

Opportunities Gained and Lost: Perceptions and Experiences of Sixth Grade Students Enrolled in a Title I Reading Class

ERIC Educational Resources Information Center

Donalson, Kathleen

2008-01-01

The purpose of this dissertation was to explore the perceptions and experiences of one class of sixth grade students enrolled in a Title I supplemental reading class. Qualitative research methods included observations, interviews, archived data, and Miscue Analysis. I examined the data through a Vygotsky constructivist perspective to provide…

Coval: Improving Alignment Quality and Variant Calling Accuracy for Next-Generation Sequencing Data

PubMed Central

Kosugi, Shunichi; Natsume, Satoshi; Yoshida, Kentaro; MacLean, Daniel; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

2013-01-01

Accurate identification of DNA polymorphisms using next-generation sequencing technology is challenging because of a high rate of sequencing error and incorrect mapping of reads to reference genomes. Currently available short read aligners and DNA variant callers suffer from these problems. We developed the Coval software to improve the quality of short read alignments. Coval is designed to minimize the incidence of spurious alignment of short reads, by filtering mismatched reads that remained in alignments after local realignment and error correction of mismatched reads. The error correction is executed based on the base quality and allele frequency at the non-reference positions for an individual or pooled sample. We demonstrated the utility of Coval by applying it to simulated genomes and experimentally obtained short-read data of rice, nematode, and mouse. Moreover, we found an unexpectedly large number of incorrectly mapped reads in ‘targeted’ alignments, where the whole genome sequencing reads had been aligned to a local genomic segment, and showed that Coval effectively eliminated such spurious alignments. We conclude that Coval significantly improves the quality of short-read sequence alignments, thereby increasing the calling accuracy of currently available tools for SNP and indel identification. Coval is available at http://sourceforge.net/projects/coval105/. PMID:24116042

Wide-Open: Accelerating public data release by automating detection of overdue datasets

PubMed Central

Poon, Hoifung; Howe, Bill

2017-01-01

Open data is a vital pillar of open science and a key enabler for reproducibility, data reuse, and novel discoveries. Enforcement of open-data policies, however, largely relies on manual efforts, which invariably lag behind the increasingly automated generation of biological data. To address this problem, we developed a general approach to automatically identify datasets overdue for public release by applying text mining to identify dataset references in published articles and parse query results from repositories to determine if the datasets remain private. We demonstrate the effectiveness of this approach on 2 popular National Center for Biotechnology Information (NCBI) repositories: Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA). Our Wide-Open system identified a large number of overdue datasets, which spurred administrators to respond directly by releasing 400 datasets in one week. PMID:28594819

Wide-Open: Accelerating public data release by automating detection of overdue datasets.

PubMed

Grechkin, Maxim; Poon, Hoifung; Howe, Bill

2017-06-01

Open data is a vital pillar of open science and a key enabler for reproducibility, data reuse, and novel discoveries. Enforcement of open-data policies, however, largely relies on manual efforts, which invariably lag behind the increasingly automated generation of biological data. To address this problem, we developed a general approach to automatically identify datasets overdue for public release by applying text mining to identify dataset references in published articles and parse query results from repositories to determine if the datasets remain private. We demonstrate the effectiveness of this approach on 2 popular National Center for Biotechnology Information (NCBI) repositories: Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA). Our Wide-Open system identified a large number of overdue datasets, which spurred administrators to respond directly by releasing 400 datasets in one week.

The Archive of the Amateur Observation Network of the International Halley Watch. Volume 1; Comet Giacobini-Zinner

NASA Technical Reports Server (NTRS)

Edberg, Stephen J. (Editor)

1996-01-01

The International Halley Watch (IHW) was organized for the purpose of gathering and archiving the most complete record of the apparition of a comet, Comet Halley (1982i = 1986 III = 1P/Halley), ever compiled. The redirection of the International Cometary Explorer (ICE), toward Comet Giacobini-Zinner (1984e = 1985 XIII = 21P/Giacobini-Zinner) prompted the initiation of a formal watch on that comet. All the data collected on P/Giacobini-Zinner and P/Halley have been published on CD-ROM in the Comet Halley Archive. This document contains a printed version of the archive data, collected by amateur astronomers, on these two comets. Volume 1 contains the Comet Giacobini-Zinner data archive and Volume 2 contains the Comet Halley archive. Both volumes include information on how to read the data in both archives, as well as a history of both comet watches (including the organizing of the network of astronomers and lessons learned from that experience).

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

PubMed

Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

2013-01-01

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

PubMed

Garcia, T I; Shen, Y; Catchen, J; Amores, A; Schartl, M; Postlethwait, J; Walter, R B

2012-01-01

For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set. Published by Elsevier Inc.

Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

PubMed

Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

2017-10-06

Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

Axe: rapid, competitive sequence read demultiplexing using a trie.

PubMed

Murray, Kevin D; Borevitz, Justin O

2018-06-01

We describe a rapid algorithm for demultiplexing DNA sequence reads with in-read indices. Axe selects the optimal index present in a sequence read, even in the presence of sequencing errors. The algorithm is able to handle combinatorial indexing, indices of differing length, and several mismatches per index sequence. Axe is implemented in C, and is used as a command-line program on Unix-like systems. Axe is available online at https://github.com/kdmurray91/axe, and is available in Debian/Ubuntu distributions of GNU/Linux as the package axe-demultiplexer. Kevin Murray axe@kdmurray.id.au. Supplementary data are available at Bioinformatics online.

RefSeq microbial genomes database: new representation and annotation strategy.

PubMed

Tatusova, Tatiana; Ciufo, Stacy; Fedorov, Boris; O'Neill, Kathleen; Tolstoy, Igor

2014-01-01

The source of the microbial genomic sequences in the RefSeq collection is the set of primary sequence records submitted to the International Nucleotide Sequence Database public archives. These can be accessed through the Entrez search and retrieval system at http://www.ncbi.nlm.nih.gov/genome. Next-generation sequencing has enabled researchers to perform genomic sequencing at rates that were unimaginable in the past. Microbial genomes can now be sequenced in a matter of hours, which has led to a significant increase in the number of assembled genomes deposited in the public archives. This huge increase in DNA sequence data presents new challenges for the annotation, analysis and visualization bioinformatics tools. New strategies have been developed for the annotation and representation of reference genomes and sequence variations derived from population studies and clinical outbreaks.

Software for pre-processing Illumina next-generation sequencing short read sequences

PubMed Central

2014-01-01

Background When compared to Sanger sequencing technology, next-generation sequencing (NGS) technologies are hindered by shorter sequence read length, higher base-call error rate, non-uniform coverage, and platform-specific sequencing artifacts. These characteristics lower the quality of their downstream analyses, e.g. de novo and reference-based assembly, by introducing sequencing artifacts and errors that may contribute to incorrect interpretation of data. Although many tools have been developed for quality control and pre-processing of NGS data, none of them provide flexible and comprehensive trimming options in conjunction with parallel processing to expedite pre-processing of large NGS datasets. Methods We developed ngsShoRT (next-generation sequencing Short Reads Trimmer), a flexible and comprehensive open-source software package written in Perl that provides a set of algorithms commonly used for pre-processing NGS short read sequences. We compared the features and performance of ngsShoRT with existing tools: CutAdapt, NGS QC Toolkit and Trimmomatic. We also compared the effects of using pre-processed short read sequences generated by different algorithms on de novo and reference-based assembly for three different genomes: Caenorhabditis elegans, Saccharomyces cerevisiae S288c, and Escherichia coli O157 H7. Results Several combinations of ngsShoRT algorithms were tested on publicly available Illumina GA II, HiSeq 2000, and MiSeq eukaryotic and bacteria genomic short read sequences with the focus on removing sequencing artifacts and low-quality reads and/or bases. Our results show that across three organisms and three sequencing platforms, trimming improved the mean quality scores of trimmed sequences. Using trimmed sequences for de novo and reference-based assembly improved assembly quality as well as assembler performance. In general, ngsShoRT outperformed comparable trimming tools in terms of trimming speed and improvement of de novo and reference-based assembly as measured by assembly contiguity and correctness. Conclusions Trimming of short read sequences can improve the quality of de novo and reference-based assembly and assembler performance. The parallel processing capability of ngsShoRT reduces trimming time and improves the memory efficiency when dealing with large datasets. We recommend combining sequencing artifacts removal, and quality score based read filtering and base trimming as the most consistent method for improving sequence quality and downstream assemblies. ngsShoRT source code, user guide and tutorial are available at http://research.bioinformatics.udel.edu/genomics/ngsShoRT/. ngsShoRT can be incorporated as a pre-processing step in genome and transcriptome assembly projects. PMID:24955109

Whole Genome Complete Resequencing of Bacillus subtilis Natto by Combining Long Reads with High-Quality Short Reads

PubMed Central

Kamada, Mayumi; Hase, Sumitaka; Sato, Kengo; Toyoda, Atsushi; Fujiyama, Asao; Sakakibara, Yasubumi

2014-01-01

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome. PMID:25329997

Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys.

PubMed

Doolittle, Russell F

2015-10-01

Lampreys and hagfish are the earliest diverging of extant vertebrates and are obvious targets for investigating the origins of complex biochemical systems found in mammals. Currently, the simplest approach for such inquiries is to search for the presence of relevant genes in whole genome sequence (WGS) assemblies. Unhappily, in the past a high-quality complete genome sequence has not been available for either lampreys or hagfish, precluding the possibility of proving gene absence. Recently, improved but still incomplete genome assemblies for two species of lamprey have been posted, and, taken together with an extensive collection of short sequences in the NCBI trace archive, they have made it possible to make reliable counts for specific gene families. Particularly, a multi-source tactic has been used to study the lamprey blood clotting system with regard to the presence and absence of genes known to occur in higher vertebrates. As was suggested in earlier studies, lampreys lack genes for coagulation factors VIII and IX, both of which are critical for the "intrinsic" clotting system and responsible for hemophilia in humans. On the other hand, they have three each of genes for factors VII and X, participants in the "extrinsic" clotting system. The strategy of using raw trace sequence "reads" together with partial WGS assemblies for lampreys can be used in studies on the early evolution of other biochemical systems in vertebrates.

Whole-genome sequencing reveals a potential causal mutation for dwarfism in the Miniature Shetland pony.

PubMed

Metzger, Julia; Gast, Alana Christina; Schrimpf, Rahel; Rau, Janina; Eikelberg, Deborah; Beineke, Andreas; Hellige, Maren; Distl, Ottmar

2017-04-01

The Miniature Shetland pony represents a horse breed with an extremely small body size. Clinical examination of a dwarf Miniature Shetland pony revealed a lowered size at the withers, malformed skull and brachygnathia superior. Computed tomography (CT) showed a shortened maxilla and a cleft of the hard and soft palate which protruded into the nasal passage leading to breathing difficulties. Pathological examination confirmed these findings but did not reveal histopathological signs of premature ossification in limbs or cranial sutures. Whole-genome sequencing of this dwarf Miniature Shetland pony and comparative sequence analysis using 26 reference equids from NCBI Sequence Read Archive revealed three probably damaging missense variants which could be exclusively found in the affected foal. Validation of these three missense mutations in 159 control horses from different horse breeds and five donkeys revealed only the aggrecan (ACAN)-associated g.94370258G>C variant as homozygous wild-type in all control samples. The dwarf Miniature Shetland pony had the homozygous mutant genotype C/C of the ACAN:g.94370258G>C variant and the normal parents were heterozygous G/C. An unaffected full sib and 3/5 unaffected half-sibs were heterozygous G/C for the ACAN:g.94370258G>C variant. In summary, we could demonstrate a dwarf phenotype in a miniature pony breed perfectly associated with a missense mutation within the ACAN gene.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

PubMed Central

Ardui, Simon; Ameur, Adam; Vermeesch, Joris R; Hestand, Matthew S

2018-01-01

Abstract Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing. PMID:29401301

ReadXplorer—visualization and analysis of mapped sequences

PubMed Central

Hilker, Rolf; Stadermann, Kai Bernd; Doppmeier, Daniel; Kalinowski, Jörn; Stoye, Jens; Straube, Jasmin; Winnebald, Jörn; Goesmann, Alexander

2014-01-01

Motivation: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. Results: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion–insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. Availability and implementation: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. Contact: rhilker@mikrobio.med.uni-giessen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24790157

A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive.

PubMed

Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

2016-01-01

Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms.

A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive

PubMed Central

Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

2016-01-01

Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms. PMID:27517583

Detection of viral sequences in archival spinal cords from fatal cases of poliomyelitis in 1951-1952.

PubMed

Rekand, Tiina; Male, Rune; Myking, Andreas O; Nygaard, Svein J T; Aarli, Johan A; Haarr, Lars; Langeland, Nina

2003-12-01

Poliovirus (PV) subjected to genetic characterization is often isolated from faecal carriage. Such virus is not necessarily identical to the virus causing paralytic disease since genetic modifications may occur during replication outside the nervous system. We have searched for poliovirus genomes in the 14 fatal cases occurring during the last epidemics in Norway in 1951-1952. A method was developed for isolation and analysis of poliovirus RNA from formalin-fixed and paraffin-embedded archival tissue. RNA was purified by incubation with Chelex-100 and heating followed by treatment with the proteinase K and chloroform extraction. Viral sequences were amplified by a reverse transcriptase-polymerase chain reaction (RT-PCR), the products subjected to TA cloning and sequenced. RNA from the beta-actin gene, as a control, was identified in 13 cases, while sequences specific for poliovirus were achieved in 11 cases. The sequences from the 2C region of poliovirus were rather conserved while those in the 5'-untranslated region were variable. The developed method should be suitable also for other genetic studies of old archival material.

What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain u...

RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA

PubMed Central

Wright, Imogen A.; Travers, Simon A.

2014-01-01

The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. PMID:24861618

Collecting, Preserving & Sharing Information in Micronesia. Proceedings of the Annual Pacific Islands Association of Libraries and Archives Conference (3rd, Saipan, Northern Mariana Islands, October 13-15, 1993).

ERIC Educational Resources Information Center

Pacific Islands Association of Libraries and Archives, Guam.

Participants from Washington, Hawaii, Majuro, Palau, Guam and other points in the Northern Mariana Islands came together to share information relating to the functions of libraries and archives as information banks and as preservers of the cultural heritage of Micronesia. Papers presented were: (1) "Reading Motivation in the Pacific"…

extendFromReads

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Kelly P.

2013-10-03

This package assists in genome assembly. extendFromReads takes as input a set of Illumina (eg, MiSeq) DNA sequencing reads, a query seed sequence and a direction to extend the seed. The algorithm collects all seed-- ]matching reads (flipping reverse-- ]orientation hits), trims off the seed and additional sequence in the other direction, sorts the remaining sequences alphabetically, and prints them aligned without gaps from the point of seed trimming. This produces a visual display distinguishing the flanks of multi- ]copy seeds. A companion script hitMates.pl collects the mates of seed-- ]hi]ng reads, whose alignment reveals longer extensions from the seed.more » The collect/trim/sort strategy was made iterative and scaled up in the script denovo.pl, for de novo contig assembly. An index is pre-- ]built using indexReads.pl that for each unique 21-- ]mer found in all the reads, records its gfate h of extension (whether extendable, blocked by low coverage, or blocked by branching after a duplicated sequence) and other characteristics. Importantly, denovo.pl records all branchings that follow a branching contig endpoint, providing contig- ]extension information« less

52nd Yearbook of the National Reading Conference (Miami, Florida, December 4-7, 2002)

ERIC Educational Resources Information Center

Fairbanks, Colleen M., Ed.; Worthy, Jo, Ed.; Maloch, Beth, Ed.; Hoffman, James V., Ed.; Schallert, Diane L., Ed.

2003-01-01

The National Reading Conference (NRC) Yearbook represents an archive of conference reports that have undergone the rigorous review that research demands, as well as an indicator of topics, ideas and concerns that occupied participants during the annual conference. With this 52nd volume of the Yearbook, the editors hope the reader finds a broad…

36 CFR 1250.12 - What types of records are available in NARA's FOIA Reading Room?

Code of Federal Regulations, 2014 CFR

2014-07-01

... 36 Parks, Forests, and Public Property 3 2014-07-01 2014-07-01 false What types of records are available in NARA's FOIA Reading Room? 1250.12 Section 1250.12 Parks, Forests, and Public Property NATIONAL ARCHIVES AND RECORDS ADMINISTRATION PUBLIC AVAILABILITY AND USE PUBLIC AVAILABILITY AND USE OF FEDERAL RECORDS General Information About Freedom...

36 CFR 1250.12 - What types of records are available in NARA's FOIA Reading Room?

Code of Federal Regulations, 2012 CFR

2012-07-01

... 36 Parks, Forests, and Public Property 3 2012-07-01 2012-07-01 false What types of records are available in NARA's FOIA Reading Room? 1250.12 Section 1250.12 Parks, Forests, and Public Property NATIONAL ARCHIVES AND RECORDS ADMINISTRATION PUBLIC AVAILABILITY AND USE PUBLIC AVAILABILITY AND USE OF FEDERAL RECORDS General Information About Freedom...

The Essential Component in DNA-Based Information Storage System: Robust Error-Tolerating Module

PubMed Central

Yim, Aldrin Kay-Yuen; Yu, Allen Chi-Shing; Li, Jing-Woei; Wong, Ada In-Chun; Loo, Jacky F. C.; Chan, King Ming; Kong, S. K.; Yip, Kevin Y.; Chan, Ting-Fung

2014-01-01

The size of digital data is ever increasing and is expected to grow to 40,000 EB by 2020, yet the estimated global information storage capacity in 2011 is <300 EB, indicating that most of the data are transient. DNA, as a very stable nano-molecule, is an ideal massive storage device for long-term data archive. The two most notable illustrations are from Church et al. and Goldman et al., whose approaches are well-optimized for most sequencing platforms – short synthesized DNA fragments without homopolymer. Here, we suggested improvements on error handling methodology that could enable the integration of DNA-based computational process, e.g., algorithms based on self-assembly of DNA. As a proof of concept, a picture of size 438 bytes was encoded to DNA with low-density parity-check error-correction code. We salvaged a significant portion of sequencing reads with mutations generated during DNA synthesis and sequencing and successfully reconstructed the entire picture. A modular-based programing framework – DNAcodec with an eXtensible Markup Language-based data format was also introduced. Our experiments demonstrated the practicability of long DNA message recovery with high error tolerance, which opens the field to biocomputing and synthetic biology. PMID:25414846

Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses

PubMed Central

Pightling, Arthur W.; Petronella, Nicholas; Pagotto, Franco

2014-01-01

The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results. PMID:25144537

MeCorS: Metagenome-enabled error correction of single cell sequencing reads

DOE PAGES

Bremges, Andreas; Singer, Esther; Woyke, Tanja; ...

2016-03-15

Here we present a new tool, MeCorS, to correct chimeric reads and sequencing errors in Illumina data generated from single amplified genomes (SAGs). It uses sequence information derived from accompanying metagenome sequencing to accurately correct errors in SAG reads, even from ultra-low coverage regions. In evaluations on real data, we show that MeCorS outperforms BayesHammer, the most widely used state-of-the-art approach. MeCorS performs particularly well in correcting chimeric reads, which greatly improves both accuracy and contiguity of de novo SAG assemblies.

Meeting the challenges of non-referenced genome assembly from short-read sequence data

Treesearch

M. Parks; A. Liston; R. Cronn

2010-01-01

Massively parallel sequencing technologies (MPST) offer unprecedented opportunities for novel sequencing projects. MPST, while offering tremendous sequencing capacity, are typically most effective in resequencing projects (as opposed to the sequencing of novel genomes) due to the fact that sequence is returned in relatively short reads. Nonetheless, there is great...

A computational method for estimating the PCR duplication rate in DNA and RNA-seq experiments.

PubMed

Bansal, Vikas

2017-03-14

PCR amplification is an important step in the preparation of DNA sequencing libraries prior to high-throughput sequencing. PCR amplification introduces redundant reads in the sequence data and estimating the PCR duplication rate is important to assess the frequency of such reads. Existing computational methods do not distinguish PCR duplicates from "natural" read duplicates that represent independent DNA fragments and therefore, over-estimate the PCR duplication rate for DNA-seq and RNA-seq experiments. In this paper, we present a computational method to estimate the average PCR duplication rate of high-throughput sequence datasets that accounts for natural read duplicates by leveraging heterozygous variants in an individual genome. Analysis of simulated data and exome sequence data from the 1000 Genomes project demonstrated that our method can accurately estimate the PCR duplication rate on paired-end as well as single-end read datasets which contain a high proportion of natural read duplicates. Further, analysis of exome datasets prepared using the Nextera library preparation method indicated that 45-50% of read duplicates correspond to natural read duplicates likely due to fragmentation bias. Finally, analysis of RNA-seq datasets from individuals in the 1000 Genomes project demonstrated that 70-95% of read duplicates observed in such datasets correspond to natural duplicates sampled from genes with high expression and identified outlier samples with a 2-fold greater PCR duplication rate than other samples. The method described here is a useful tool for estimating the PCR duplication rate of high-throughput sequence datasets and for assessing the fraction of read duplicates that correspond to natural read duplicates. An implementation of the method is available at https://github.com/vibansal/PCRduplicates .

ORFer--retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files.

PubMed

Büssow, Konrad; Hoffmann, Steve; Sievert, Volker

2002-12-19

Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information.

Short-Read Sequencing for Genomic Analysis of the Brown Rot Fungus Fibroporia radiculosa

Treesearch

J. D. Tang; A. D. Perkins; T. S. Sonstegard; S. G. Schroeder; S. C. Burgess; S. V. Diehl

2012-01-01

The feasibility of short-read sequencing for genomic analysis was demonstrated for Fibroporia radiculosa, a copper-tolerant fungus that causes brown rot decay of wood. The effect of read quality on genomic assembly was assessed by filtering Illumina GAIIx reads from a single run of a paired-end library (75-nucleotide read length and 300-bp fragment...

Types and Sequences of Self-Regulated Reading of Low-Achieving Adolescents in Relation to Reading Task Achievement

ERIC Educational Resources Information Center

de Milliano, Ilona; van Gelderen, Amos; Sleegers, Peter

2016-01-01

This study examines the relationship between types and sequences of self-regulated reading activities in task-oriented reading with quality of task achievement of 51 low-achieving adolescents (Grade 8). The study used think aloud combined with video observations to analyse the students' approach of a content-area reading task in the stages of…

Rcorrector: efficient and accurate error correction for Illumina RNA-seq reads.

PubMed

Song, Li; Florea, Liliana

2015-01-01

Next-generation sequencing of cellular RNA (RNA-seq) is rapidly becoming the cornerstone of transcriptomic analysis. However, sequencing errors in the already short RNA-seq reads complicate bioinformatics analyses, in particular alignment and assembly. Error correction methods have been highly effective for whole-genome sequencing (WGS) reads, but are unsuitable for RNA-seq reads, owing to the variation in gene expression levels and alternative splicing. We developed a k-mer based method, Rcorrector, to correct random sequencing errors in Illumina RNA-seq reads. Rcorrector uses a De Bruijn graph to compactly represent all trusted k-mers in the input reads. Unlike WGS read correctors, which use a global threshold to determine trusted k-mers, Rcorrector computes a local threshold at every position in a read. Rcorrector has an accuracy higher than or comparable to existing methods, including the only other method (SEECER) designed for RNA-seq reads, and is more time and memory efficient. With a 5 GB memory footprint for 100 million reads, it can be run on virtually any desktop or server. The software is available free of charge under the GNU General Public License from https://github.com/mourisl/Rcorrector/.

Re-examination of population structure and phylogeography of hawksbill turtles in the wider Caribbean using longer mtDNA sequences.

PubMed

Leroux, Robin A; Dutton, Peter H; Abreu-Grobois, F Alberto; Lagueux, Cynthia J; Campbell, Cathi L; Delcroix, Eric; Chevalier, Johan; Horrocks, Julia A; Hillis-Starr, Zandy; Troëng, Sebastian; Harrison, Emma; Stapleton, Seth

2012-01-01

Management of the critically endangered hawksbill turtle in the Wider Caribbean (WC) has been hampered by knowledge gaps regarding stock structure. We carried out a comprehensive stock structure re-assessment of 11 WC hawksbill rookeries using longer mtDNA sequences, larger sample sizes (N = 647), and additional rookeries compared to previous surveys. Additional variation detected by 740 bp sequences between populations allowed us to differentiate populations such as Barbados-Windward and Guadeloupe (F (st) = 0.683, P < 0.05) that appeared genetically indistinguishable based on shorter 380 bp sequences. POWSIM analysis showed that longer sequences improved power to detect population structure and that when N < 30, increasing the variation detected was as effective in increasing power as increasing sample size. Geographic patterns of genetic variation suggest a model of periodic long-distance colonization coupled with region-wide dispersal and subsequent secondary contact within the WC. Mismatch analysis results for individual clades suggest a general population expansion in the WC following a historic bottleneck about 100 000-300 000 years ago. We estimated an effective female population size (N (ef)) of 6000-9000 for the WC, similar to the current estimated numbers of breeding females, highlighting the importance of these regional rookeries to maintaining genetic diversity in hawksbills. Our results provide a basis for standardizing future work to 740 bp sequence reads and establish a more complete baseline for determining stock boundaries in this migratory marine species. Finally, our findings illustrate the value of maintaining an archive of specimens for re-analysis as new markers become available.

Haematobia irritans dataset of raw sequence reads from Illumina and Pac Bio sequencing of genomic DNA

USDA-ARS?s Scientific Manuscript database

The genome of the horn fly, Haematobia irritans, was sequenced using Illumina- and Pac Bio-based protocols. Following quality filtering, the raw reads have been deposited at NCBI under the BioProject and BioSample accession numbers PRJNA30967 and SAMN07830356, respectively. The Illumina reads are un...

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

PubMed Central

Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J.; Rohan, Thomas; Ben-Dov, Iddo Z.

2017-01-01

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens. PMID:28335433

Metagenome assembly through clustering of next-generation sequencing data using protein sequences.

PubMed

Sim, Mikang; Kim, Jaebum

2015-02-01

The study of environmental microbial communities, called metagenomics, has gained a lot of attention because of the recent advances in next-generation sequencing (NGS) technologies. Microbes play a critical role in changing their environments, and the mode of their effect can be solved by investigating metagenomes. However, the difficulty of metagenomes, such as the combination of multiple microbes and different species abundance, makes metagenome assembly tasks more challenging. In this paper, we developed a new metagenome assembly method by utilizing protein sequences, in addition to the NGS read sequences. Our method (i) builds read clusters by using mapping information against available protein sequences, and (ii) creates contig sequences by finding consensus sequences through probabilistic choices from the read clusters. By using simulated NGS read sequences from real microbial genome sequences, we evaluated our method in comparison with four existing assembly programs. We found that our method could generate relatively long and accurate metagenome assemblies, indicating that the idea of using protein sequences, as a guide for the assembly, is promising. Copyright © 2015 Elsevier B.V. All rights reserved.

Modeling read counts for CNV detection in exome sequencing data.

PubMed

Love, Michael I; Myšičková, Alena; Sun, Ruping; Kalscheuer, Vera; Vingron, Martin; Haas, Stefan A

2011-11-08

Varying depth of high-throughput sequencing reads along a chromosome makes it possible to observe copy number variants (CNVs) in a sample relative to a reference. In exome and other targeted sequencing projects, technical factors increase variation in read depth while reducing the number of observed locations, adding difficulty to the problem of identifying CNVs. We present a hidden Markov model for detecting CNVs from raw read count data, using background read depth from a control set as well as other positional covariates such as GC-content. The model, exomeCopy, is applied to a large chromosome X exome sequencing project identifying a list of large unique CNVs. CNVs predicted by the model and experimentally validated are then recovered using a cross-platform control set from publicly available exome sequencing data. Simulations show high sensitivity for detecting heterozygous and homozygous CNVs, outperforming normalization and state-of-the-art segmentation methods.

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets.

PubMed

Hosseini, Parsa; Tremblay, Arianne; Matthews, Benjamin F; Alkharouf, Nadim W

2010-07-02

The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially designed to translate sequence data in a CASAVA-build into functional annotations while producing corresponding gene expression measurements. Achieving such analysis is executed in an ultrafast and highly efficient manner, whether the analysis be a single-read or paired-end sequencing experiment. TASE is a user-friendly and freely available application, allowing rapid analysis and annotation of any given Illumina Solexa sequencing dataset with ease.

Genome Sequencing and Assembly by Long Reads in Plants

PubMed Central

Li, Changsheng; Lin, Feng; An, Dong; Huang, Ruidong

2017-01-01

Plant genomes generated by Sanger and Next Generation Sequencing (NGS) have provided insight into species diversity and evolution. However, Sanger sequencing is limited in its applications due to high cost, labor intensity, and low throughput, while NGS reads are too short to resolve abundant repeats and polyploidy, leading to incomplete or ambiguous assemblies. The advent and improvement of long-read sequencing by Third Generation Sequencing (TGS) methods such as PacBio and Nanopore have shown promise in producing high-quality assemblies for complex genomes. Here, we review the development of sequencing, introducing the application as well as considerations of experimental design in TGS of plant genomes. We also introduce recent revolutionary scaffolding technologies including BioNano, Hi-C, and 10× Genomics. We expect that the informative guidance for genome sequencing and assembly by long reads will benefit the initiation of scientists’ projects. PMID:29283420

Probability of coding of a DNA sequence: an algorithm to predict translated reading frames from their thermodynamic characteristics.

PubMed Central

Tramontano, A; Macchiato, M F

1986-01-01

An algorithm to determine the probability that a reading frame codifies for a protein is presented. It is based on the results of our previous studies on the thermodynamic characteristics of a translated reading frame. We also develop a prediction procedure to distinguish between coding and non-coding reading frames. The procedure is based on the characteristics of the putative product of the DNA sequence and not on periodicity characteristics of the sequence, so the prediction is not biased by the presence of overlapping translated reading frames or by the presence of translated reading frames on the complementary DNA strand. PMID:3753761

HSA: a heuristic splice alignment tool.

PubMed

Bu, Jingde; Chi, Xuebin; Jin, Zhong

2013-01-01

RNA-Seq methodology is a revolutionary transcriptomics sequencing technology, which is the representative of Next generation Sequencing (NGS). With the high throughput sequencing of RNA-Seq, we can acquire much more information like differential expression and novel splice variants from deep sequence analysis and data mining. But the short read length brings a great challenge to alignment, especially when the reads span two or more exons. A two steps heuristic splice alignment tool is generated in this investigation. First, map raw reads to reference with unspliced aligner--BWA; second, split initial unmapped reads into three equal short reads (seeds), align each seed to the reference, filter hits, search possible split position of read and extend hits to a complete match. Compare with other splice alignment tools like SOAPsplice and Tophat2, HSA has a better performance in call rate and efficiency, but its results do not as accurate as the other software to some extent. HSA is an effective spliced aligner of RNA-Seq reads mapping, which is available at https://github.com/vlcc/HSA.

An optimized protocol for generation and analysis of Ion Proton sequencing reads for RNA-Seq.

PubMed

Yuan, Yongxian; Xu, Huaiqian; Leung, Ross Ka-Kit

2016-05-26

Previous studies compared running cost, time and other performance measures of popular sequencing platforms. However, comprehensive assessment of library construction and analysis protocols for Proton sequencing platform remains unexplored. Unlike Illumina sequencing platforms, Proton reads are heterogeneous in length and quality. When sequencing data from different platforms are combined, this can result in reads with various read length. Whether the performance of the commonly used software for handling such kind of data is satisfactory is unknown. By using universal human reference RNA as the initial material, RNaseIII and chemical fragmentation methods in library construction showed similar result in gene and junction discovery number and expression level estimated accuracy. In contrast, sequencing quality, read length and the choice of software affected mapping rate to a much larger extent. Unspliced aligner TMAP attained the highest mapping rate (97.27 % to genome, 86.46 % to transcriptome), though 47.83 % of mapped reads were clipped. Long reads could paradoxically reduce mapping in junctions. With reference annotation guide, the mapping rate of TopHat2 significantly increased from 75.79 to 92.09 %, especially for long (>150 bp) reads. Sailfish, a k-mer based gene expression quantifier attained highly consistent results with that of TaqMan array and highest sensitivity. We provided for the first time, the reference statistics of library preparation methods, gene detection and quantification and junction discovery for RNA-Seq by the Ion Proton platform. Chemical fragmentation performed equally well with the enzyme-based one. The optimal Ion Proton sequencing options and analysis software have been evaluated.

RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA.

PubMed

Wright, Imogen A; Travers, Simon A

2014-07-01

The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Detection of microRNAs in color space.

PubMed

Marco, Antonio; Griffiths-Jones, Sam

2012-02-01

Deep sequencing provides inexpensive opportunities to characterize the transcriptional diversity of known genomes. The AB SOLiD technology generates millions of short sequencing reads in color-space; that is, the raw data is a sequence of colors, where each color represents 2 nt and each nucleotide is represented by two consecutive colors. This strategy is purported to have several advantages, including increased ability to distinguish sequencing errors from polymorphisms. Several programs have been developed to map short reads to genomes in color space. However, a number of previously unexplored technical issues arise when using SOLiD technology to characterize microRNAs. Here we explore these technical difficulties. First, since the sequenced reads are longer than the biological sequences, every read is expected to contain linker fragments. The color-calling error rate increases toward the 3(') end of the read such that recognizing the linker sequence for removal becomes problematic. Second, mapping in color space may lead to the loss of the first nucleotide of each read. We propose a sequential trimming and mapping approach to map small RNAs. Using our strategy, we reanalyze three published insect small RNA deep sequencing datasets and characterize 22 new microRNAs. A bash shell script to perform the sequential trimming and mapping procedure, called SeqTrimMap, is available at: http://www.mirbase.org/tools/seqtrimmap/ antonio.marco@manchester.ac.uk Supplementary data are available at Bioinformatics online.

SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information

PubMed Central

2014-01-01

Background The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. Results Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. Conclusions The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. PMID:24950923

36 CFR § 1250.12 - What types of records are available in NARA's FOIA Reading Room?

Code of Federal Regulations, 2013 CFR

2013-07-01

... 36 Parks, Forests, and Public Property 3 2013-07-01 2012-07-01 true What types of records are available in NARA's FOIA Reading Room? § 1250.12 Section § 1250.12 Parks, Forests, and Public Property NATIONAL ARCHIVES AND RECORDS ADMINISTRATION PUBLIC AVAILABILITY AND USE PUBLIC AVAILABILITY AND USE OF FEDERAL RECORDS General Information About...

Differences in Reading and Math Achievement among Students Who Are Hispanic, Limited English Proficient, or White: A Multi-Year Study

ERIC Educational Resources Information Center

Rojas-LeBouef, Ana M.

2010-01-01

Purpose. The purpose of this study was to examine differences in academic achievement among students who were Hispanic, Limited English Proficient (LEP), or White, using archival data from the Texas Education Agency's (TEA) Academic Excellence Indicator System (AEIS). Data examined were fifth grade reading and math passing rates from the 1993…

Long Reads: their Purpose and Place.

PubMed

Pollard, Martin O; Gurdasani, Deepti; Mentzer, Alexander J; Porter, Tarryn; Sandhu, Manjinder S

2018-05-14

In recent years long read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing (NGS), the cost of sequencing using long read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterising genomes at high resolution. In this article, we will endeavour to present an introduction to long read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful; and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

LoRTE: Detecting transposon-induced genomic variants using low coverage PacBio long read sequences.

PubMed

Disdero, Eric; Filée, Jonathan

2017-01-01

Population genomic analysis of transposable elements has greatly benefited from recent advances of sequencing technologies. However, the short size of the reads and the propensity of transposable elements to nest in highly repeated regions of genomes limits the efficiency of bioinformatic tools when Illumina or 454 technologies are used. Fortunately, long read sequencing technologies generating read length that may span the entire length of full transposons are now available. However, existing TE population genomic softwares were not designed to handle long reads and the development of new dedicated tools is needed. LoRTE is the first tool able to use PacBio long read sequences to identify transposon deletions and insertions between a reference genome and genomes of different strains or populations. Tested against simulated and genuine Drosophila melanogaster PacBio datasets, LoRTE appears to be a reliable and broadly applicable tool to study the dynamic and evolutionary impact of transposable elements using low coverage, long read sequences. LoRTE is an efficient and accurate tool to identify structural genomic variants caused by TE insertion or deletion. LoRTE is available for download at http://www.egce.cnrs-gif.fr/?p=6422.

The Archive of the Amateur Observation Network of the International Halley Watch. Volume 2; Comet Halley

NASA Technical Reports Server (NTRS)

Edberg, Stephen J. (Editor)

1996-01-01

The International Halley Watch (IHW) was organized for the purpose of gathering and archiving the most complete record of the apparition of a comet, Halley's Comet (1982i = 1986 III = 1P/Halley), ever compiled. The redirection of the International Sun-Earth Explorer 3 (ISEE-3) spacecraft, subsequently renamed the International Cometary Explorer (ICE), toward Comet Giacobini- Zinner (1984e = 1985 XIII = 21P/Giacobini-Zinner) prompted the initiation of a formal watch on that comet. All the data collected on P/Giacobini-Zinner and P/Halley have been published on CD-ROM in the Comet Halley Archive. This document contains a printed version of the archive data, collected by amateur astronomers, on these two comets. Volume 1 contains the Comet Giacobini-Zinner data archive and Volume 2 contains the Comet Halley archive. Both volumes include information on how to read the data in both archives, as well as a history of both comet watches (including the organizing of the network of astronomers and lessons learned from that experience).

The Archive of the Amateur Observation Network of the International Halley Watch. Volume 1; Comet Giacobini-Zinner

NASA Technical Reports Server (NTRS)

Edberg, Stephen J. (Editor)

1966-01-01

The International Halley Watch (IHW) was organized for the purpose of gathering and archiving the most complete record of the apparition of a comet, Halley's Comet (1982i = 1986 III = 1P/Halley), ever compiled. The redirection of the International Sun-Earth Explorer 3 (ISEE-3) spacecraft, subsequently renamed the International Cometary Explorer (ICE), toward Comet Giacobini-Zinner (1984e = 1985 XIII = 21P/Giacobini-Zinner) prompted the initiation of a formal watch on that comet. All the data collected on P/Giacobini-Zinner and P/Halley have been published on CD-ROM in the Comet Halley Archive. This document contains a printed version of the archive data, collected by amateur astronomers, on these two comets. Volume 1 contains the Comet Giacobini-Zinner data archive and Volume 2 contains the Comet Halley archive. Both volumes include information on how to read the data in both archives, as well as a history of both comet watches (including the organizing of the network of astronomers and lessons learned from that experience).

Sequence History Update Tool

NASA Technical Reports Server (NTRS)

Khanampompan, Teerapat; Gladden, Roy; Fisher, Forest; DelGuercio, Chris

2008-01-01

The Sequence History Update Tool performs Web-based sequence statistics archiving for Mars Reconnaissance Orbiter (MRO). Using a single UNIX command, the software takes advantage of sequencing conventions to automatically extract the needed statistics from multiple files. This information is then used to populate a PHP database, which is then seamlessly formatted into a dynamic Web page. This tool replaces a previous tedious and error-prone process of manually editing HTML code to construct a Web-based table. Because the tool manages all of the statistics gathering and file delivery to and from multiple data sources spread across multiple servers, there is also a considerable time and effort savings. With the use of The Sequence History Update Tool what previously took minutes is now done in less than 30 seconds, and now provides a more accurate archival record of the sequence commanding for MRO.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dolan, Daniel H.; Ao, Tommy

The Sandia Data Archive (SDA) format is a specific implementation of the HDF5 (Hierarchal Data Format version 5) standard. The format was developed for storing data in a universally accessible manner. SDA files may contain one or more data records, each associated with a distinct text label. Primitive records provide basic data storage, while compound records support more elaborate grouping. External records allow text/binary files to be carried inside an archive and later recovered. This report documents version 1.0 of the SDA standard. The information provided here is sufficient for reading from and writing to an archive. Although the formatmore » was original designed for use in MATLAB, broader use is encouraged.« less

Metagenomic investigation of the microbial diversity in a chrysotile asbestos mine pit pond, Lowell, Vermont, USA.

PubMed

Driscoll, Heather E; Vincent, James J; English, Erika L; Dolci, Elizabeth D

2016-12-01

Here we report on a metagenomics investigation of the microbial diversity in a serpentine-hosted aquatic habitat created by chrysotile asbestos mining activity at the Vermont Asbestos Group (VAG) Mine in northern Vermont, USA. The now-abandoned VAG Mine on Belvidere Mountain in the towns of Eden and Lowell includes three open-pit quarries, a flooded pit, mill buildings, roads, and > 26 million metric tons of eroding mine waste that contribute alkaline mine drainage to the surrounding watershed. Metagenomes and water chemistry originated from aquatic samples taken at three depths (0.5 m, 3.5 m, and 25 m) along the water column at three distinct, offshore sites within the mine's flooded pit (near 44°46'00.7673″, - 72°31'36.2699″; UTM NAD 83 Zone 18 T 0695720 E, 4960030 N). Whole metagenome shotgun Illumina paired-end sequences were quality trimmed and analyzed based on a translated nucleotide search of NCBI-NR protein database and lowest common ancestor taxonomic assignments. Our results show strata within the pit pond water column can be distinguished by taxonomic composition and distribution, pH, temperature, conductivity, light intensity, and concentrations of dissolved oxygen. At the phylum level, metagenomes from 0.5 m and 3.5 m contained a similar distribution of taxa and were dominated by Actinobacteria (46% and 53% of reads, respectively), Proteobacteria (45% and 38%, respectively), and Bacteroidetes (7% in both). The metagenomes from 25 m showed a greater diversity of phyla and a different distribution of reads than the two upper strata: Proteobacteria (60%), Actinobacteria (18%), Planctomycetes, (10%), Bacteroidetes (5%) and Cyanobacteria (2.5%), Armatimonadetes (< 1%), Verrucomicrobia (< 1%), Firmicutes (< 1%), and Nitrospirae (< 1%). Raw metagenome sequence data from each sample reside in NCBI's Short Read Archive (SRA ID: SRP056095) and are accessible through NCBI BioProject PRJNA277916.

RNA-seq Analysis of Early Hepatic Response to Handling and Confinement Stress in Rainbow Trout

PubMed Central

Liu, Sixin; Gao, Guangtu; Palti, Yniv; Cleveland, Beth M.; Weber, Gregory M.; Rexroad, Caird E.

2014-01-01

Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R2 = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database. Accession Numbers Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881. Customized Perl Scripts All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author. PMID:24558395

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver.

PubMed

Wymant, Chris; Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ong, Swee Hoe; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Berkhout, Ben; Cornelissen, Marion; Kellam, Paul; Reiss, Peter; Fraser, Christophe

2018-01-01

Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.

Stroma Based Prognosticators Incorporating Differences between African and European Americans

DTIC Science & Technology

2017-10-01

amenable to bisulfite sequencing of more than a few genes. Exploiting the recent three-fold reduction in the cost of sequencing per read , we developed oligo...cards. The ability of the HiSeq 4000 to obtain about three times as many reads as the HiSeq2500, at the same price, means we can stay on track, though...capture, and sequencing (Table 2). We obtain tens of millions of mapped deduplicated reads per sample, while using only 5% of a sequencing lane per sample

Hybrid-denovo: a de novo OTU-picking pipeline integrating single-end and paired-end 16S sequence tags.

PubMed

Chen, Xianfeng; Johnson, Stephen; Jeraldo, Patricio; Wang, Junwen; Chia, Nicholas; Kocher, Jean-Pierre A; Chen, Jun

2018-03-01

Illumina paired-end sequencing has been increasingly popular for 16S rRNA gene-based microbiota profiling. It provides higher phylogenetic resolution than single-end reads due to a longer read length. However, the reverse read (R2) often has significant low base quality, and a large proportion of R2s will be discarded after quality control, resulting in a mixture of paired-end and single-end reads. A typical 16S analysis pipeline usually processes either paired-end or single-end reads but not a mixture. Thus, the quantification accuracy and statistical power will be reduced due to the loss of a large amount of reads. As a result, rare taxa may not be detectable with the paired-end approach, or low taxonomic resolution will result in a single-end approach. To have both the higher phylogenetic resolution provided by paired-end reads and the higher sequence coverage by single-end reads, we propose a novel OTU-picking pipeline, hybrid-denovo, that can process a hybrid of single-end and paired-end reads. Using high-quality paired-end reads as a gold standard, we show that hybrid-denovo achieved the highest correlation with the gold standard and performed better than the approaches based on paired-end or single-end reads in terms of quantifying the microbial diversity and taxonomic abundances. By applying our method to a rheumatoid arthritis (RA) data set, we demonstrated that hybrid-denovo captured more microbial diversity and identified more RA-associated taxa than a paired-end or single-end approach. Hybrid-denovo utilizes both paired-end and single-end 16S sequencing reads and is recommended for 16S rRNA gene targeted paired-end sequencing data.

Issues in Electronic Publishing.

ERIC Educational Resources Information Center

Meadow, Charles T.

1997-01-01

Discusses issues related to electronic publishing. Topics include writing; reading; production, distribution, and commerce; copyright and ownership of intellectual property; archival storage; technical obsolescence; control of content; equality of access; and cultural changes. (Author/LRW)

Illuminating the Black Box of Genome Sequence Assembly: A Free Online Tool to Introduce Students to Bioinformatics

ERIC Educational Resources Information Center

Taylor, D. Leland; Campbell, A. Malcolm; Heyer, Laurie J.

2013-01-01

Next-generation sequencing technologies have greatly reduced the cost of sequencing genomes. With the current sequencing technology, a genome is broken into fragments and sequenced, producing millions of "reads." A computer algorithm pieces these reads together in the genome assembly process. PHAST is a set of online modules…

Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool.

PubMed

Jérôme, Mariette; Noirot, Céline; Klopp, Christophe

2011-05-26

Roche 454 pyrosequencing platform is often considered the most versatile of the Next Generation Sequencing technology platforms, permitting the sequencing of large genomes, the analysis of variations or the study of transcriptomes. A recent reported bias leads to the production of multiple reads for a unique DNA fragment in a random manner within a run. This bias has a direct impact on the quality of the measurement of the representation of the fragments using the reads. Other cleaning steps are usually performed on the reads before assembly or alignment. PyroCleaner is a software module intended to clean 454 pyrosequencing reads in order to ease the assembly process. This program is a free software and is distributed under the terms of the GNU General Public License as published by the Free Software Foundation. It implements several filters using criteria such as read duplication, length, complexity, base-pair quality and number of undetermined bases. It also permits to clean flowgram files (.sff) of paired-end sequences generating on one hand validated paired-ends file and the other hand single read file. Read cleaning has always been an important step in sequence analysis. The pyrocleaner python module is a Swiss knife dedicated to 454 reads cleaning. It includes commonly used filters as well as specialised ones such as duplicated read removal and paired-end read verification.

Development and transferability of black and red raspberry microsatellite markers from short-read sequences

USDA-ARS?s Scientific Manuscript database

The advent of next-generation sequencing technologies has been a boon to the cost-effective development of molecular markers, particularly in non-model species. Here, we demonstrate the efficiency of microsatellite or simple sequence repeat (SSR) marker development from short-read sequences using th...

VariantBam: filtering and profiling of next-generational sequencing data using region-specific rules.

PubMed

Wala, Jeremiah; Zhang, Cheng-Zhong; Meyerson, Matthew; Beroukhim, Rameen

2016-07-01

We developed VariantBam, a C ++ read filtering and profiling tool for use with BAM, CRAM and SAM sequencing files. VariantBam provides a flexible framework for extracting sequencing reads or read-pairs that satisfy combinations of rules, defined by any number of genomic intervals or variant sites. We have implemented filters based on alignment data, sequence motifs, regional coverage and base quality. For example, VariantBam achieved a median size reduction ratio of 3.1:1 when applied to 10 lung cancer whole genome BAMs by removing large tags and selecting for only high-quality variant-supporting reads and reads matching a large dictionary of sequence motifs. Thus VariantBam enables efficient storage of sequencing data while preserving the most relevant information for downstream analysis. VariantBam and full documentation are available at github.com/jwalabroad/VariantBam rameen@broadinstitute.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Assessing the performance of the Oxford Nanopore Technologies MinION

PubMed Central

Laver, T.; Harrison, J.; O’Neill, P.A.; Moore, K.; Farbos, A.; Paszkiewicz, K.; Studholme, D.J.

2015-01-01

The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G + C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2 kb and 1 kb respectively, while the longest single read was 98 kb. The whole length of a 5 kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data. PMID:26753127

2010 CSAF Reading List

Science.gov Websites

Speeches Archive Former AF Top 3 Viewpoints and Speeches Air Force Warrior Games 2017 Events 2018 Air Force Strategic Documents Desert Storm 25th Anniversary Observances DoD Warrior Games Portraits in Courage

2007 CSAF Reading List

Science.gov Websites

Speeches Archive Former AF Top 3 Viewpoints and Speeches Air Force Warrior Games 2017 Events 2018 Air Force Strategic Documents Desert Storm 25th Anniversary Observances DoD Warrior Games Portraits in Courage

2008 CSAF Reading List

Science.gov Websites

Speeches Archive Former AF Top 3 Viewpoints and Speeches Air Force Warrior Games 2017 Events 2018 Air Force Strategic Documents Desert Storm 25th Anniversary Observances DoD Warrior Games Portraits in Courage

2011 CSAF Reading List

Science.gov Websites

Speeches Archive Former AF Top 3 Viewpoints and Speeches Air Force Warrior Games 2017 Events 2018 Air Force Strategic Documents Desert Storm 25th Anniversary Observances DoD Warrior Games Portraits in Courage

2009 CSAF Reading List

Science.gov Websites

Speeches Archive Former AF Top 3 Viewpoints and Speeches Air Force Warrior Games 2017 Events 2018 Air Force Strategic Documents Desert Storm 25th Anniversary Observances DoD Warrior Games Portraits in Courage

Improving RNA-Seq expression estimation by modeling isoform- and exon-specific read sequencing rate.

PubMed

Liu, Xuejun; Shi, Xinxin; Chen, Chunlin; Zhang, Li

2015-10-16

The high-throughput sequencing technology, RNA-Seq, has been widely used to quantify gene and isoform expression in the study of transcriptome in recent years. Accurate expression measurement from the millions or billions of short generated reads is obstructed by difficulties. One is ambiguous mapping of reads to reference transcriptome caused by alternative splicing. This increases the uncertainty in estimating isoform expression. The other is non-uniformity of read distribution along the reference transcriptome due to positional, sequencing, mappability and other undiscovered sources of biases. This violates the uniform assumption of read distribution for many expression calculation approaches, such as the direct RPKM calculation and Poisson-based models. Many methods have been proposed to address these difficulties. Some approaches employ latent variable models to discover the underlying pattern of read sequencing. However, most of these methods make bias correction based on surrounding sequence contents and share the bias models by all genes. They therefore cannot estimate gene- and isoform-specific biases as revealed by recent studies. We propose a latent variable model, NLDMseq, to estimate gene and isoform expression. Our method adopts latent variables to model the unknown isoforms, from which reads originate, and the underlying percentage of multiple spliced variants. The isoform- and exon-specific read sequencing biases are modeled to account for the non-uniformity of read distribution, and are identified by utilizing the replicate information of multiple lanes of a single library run. We employ simulation and real data to verify the performance of our method in terms of accuracy in the calculation of gene and isoform expression. Results show that NLDMseq obtains competitive gene and isoform expression compared to popular alternatives. Finally, the proposed method is applied to the detection of differential expression (DE) to show its usefulness in the downstream analysis. The proposed NLDMseq method provides an approach to accurately estimate gene and isoform expression from RNA-Seq data by modeling the isoform- and exon-specific read sequencing biases. It makes use of a latent variable model to discover the hidden pattern of read sequencing. We have shown that it works well in both simulations and real datasets, and has competitive performance compared to popular methods. The method has been implemented as a freely available software which can be found at https://github.com/PUGEA/NLDMseq.

An efficient annotation and gene-expression derivation tool for Illumina Solexa datasets

PubMed Central

2010-01-01

Background The data produced by an Illumina flow cell with all eight lanes occupied, produces well over a terabyte worth of images with gigabytes of reads following sequence alignment. The ability to translate such reads into meaningful annotation is therefore of great concern and importance. Very easily, one can get flooded with such a great volume of textual, unannotated data irrespective of read quality or size. CASAVA, a optional analysis tool for Illumina sequencing experiments, enables the ability to understand INDEL detection, SNP information, and allele calling. To not only extract from such analysis, a measure of gene expression in the form of tag-counts, but furthermore to annotate such reads is therefore of significant value. Findings We developed TASE (Tag counting and Analysis of Solexa Experiments), a rapid tag-counting and annotation software tool specifically designed for Illumina CASAVA sequencing datasets. Developed in Java and deployed using jTDS JDBC driver and a SQL Server backend, TASE provides an extremely fast means of calculating gene expression through tag-counts while annotating sequenced reads with the gene's presumed function, from any given CASAVA-build. Such a build is generated for both DNA and RNA sequencing. Analysis is broken into two distinct components: DNA sequence or read concatenation, followed by tag-counting and annotation. The end result produces output containing the homology-based functional annotation and respective gene expression measure signifying how many times sequenced reads were found within the genomic ranges of functional annotations. Conclusions TASE is a powerful tool to facilitate the process of annotating a given Illumina Solexa sequencing dataset. Our results indicate that both homology-based annotation and tag-count analysis are achieved in very efficient times, providing researchers to delve deep in a given CASAVA-build and maximize information extraction from a sequencing dataset. TASE is specially designed to translate sequence data in a CASAVA-build into functional annotations while producing corresponding gene expression measurements. Achieving such analysis is executed in an ultrafast and highly efficient manner, whether the analysis be a single-read or paired-end sequencing experiment. TASE is a user-friendly and freely available application, allowing rapid analysis and annotation of any given Illumina Solexa sequencing dataset with ease. PMID:20598141

Designing robust watermark barcodes for multiplex long-read sequencing.

PubMed

Ezpeleta, Joaquín; Krsticevic, Flavia J; Bulacio, Pilar; Tapia, Elizabeth

2017-03-15

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed. We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process. Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark . ezpeleta@cifasis-conicet.gov.ar. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Long-read sequencing data analysis for yeasts.

PubMed

Yue, Jia-Xing; Liti, Gianni

2018-06-01

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ∼41 h to generate a complete and well-annotated genome from ∼100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

Low-Bandwidth and Non-Compute Intensive Remote Identification of Microbes from Raw Sequencing Reads

PubMed Central

Gautier, Laurent; Lund, Ole

2013-01-01

Cheap DNA sequencing may soon become routine not only for human genomes but also for practically anything requiring the identification of living organisms from their DNA: tracking of infectious agents, control of food products, bioreactors, or environmental samples. We propose a novel general approach to the analysis of sequencing data where a reference genome does not have to be specified. Using a distributed architecture we are able to query a remote server for hints about what the reference might be, transferring a relatively small amount of data. Our system consists of a server with known reference DNA indexed, and a client with raw sequencing reads. The client sends a sample of unidentified reads, and in return receives a list of matching references. Sequences for the references can be retrieved and used for exhaustive computation on the reads, such as alignment. To demonstrate this approach we have implemented a web server, indexing tens of thousands of publicly available genomes and genomic regions from various organisms and returning lists of matching hits from query sequencing reads. We have also implemented two clients: one running in a web browser, and one as a python script. Both are able to handle a large number of sequencing reads and from portable devices (the browser-based running on a tablet), perform its task within seconds, and consume an amount of bandwidth compatible with mobile broadband networks. Such client-server approaches could develop in the future, allowing a fully automated processing of sequencing data and routine instant quality check of sequencing runs from desktop sequencers. A web access is available at http://tapir.cbs.dtu.dk. The source code for a python command-line client, a server, and supplementary data are available at http://bit.ly/1aURxkc. PMID:24391826

Low-bandwidth and non-compute intensive remote identification of microbes from raw sequencing reads.

PubMed

Gautier, Laurent; Lund, Ole

2013-01-01

Cheap DNA sequencing may soon become routine not only for human genomes but also for practically anything requiring the identification of living organisms from their DNA: tracking of infectious agents, control of food products, bioreactors, or environmental samples. We propose a novel general approach to the analysis of sequencing data where a reference genome does not have to be specified. Using a distributed architecture we are able to query a remote server for hints about what the reference might be, transferring a relatively small amount of data. Our system consists of a server with known reference DNA indexed, and a client with raw sequencing reads. The client sends a sample of unidentified reads, and in return receives a list of matching references. Sequences for the references can be retrieved and used for exhaustive computation on the reads, such as alignment. To demonstrate this approach we have implemented a web server, indexing tens of thousands of publicly available genomes and genomic regions from various organisms and returning lists of matching hits from query sequencing reads. We have also implemented two clients: one running in a web browser, and one as a python script. Both are able to handle a large number of sequencing reads and from portable devices (the browser-based running on a tablet), perform its task within seconds, and consume an amount of bandwidth compatible with mobile broadband networks. Such client-server approaches could develop in the future, allowing a fully automated processing of sequencing data and routine instant quality check of sequencing runs from desktop sequencers. A web access is available at http://tapir.cbs.dtu.dk. The source code for a python command-line client, a server, and supplementary data are available at http://bit.ly/1aURxkc.

Accurate typing of short tandem repeats from genome-wide sequencing data and its applications.

PubMed

Fungtammasan, Arkarachai; Ananda, Guruprasad; Hile, Suzanne E; Su, Marcia Shu-Wei; Sun, Chen; Harris, Robert; Medvedev, Paul; Eckert, Kristin; Makova, Kateryna D

2015-05-01

Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution. © 2015 Fungtammasan et al.; Published by Cold Spring Harbor Laboratory Press.

Discovery and Annotation of Plant Endogenous Target Mimicry Sequences from Public Transcriptome Libraries: A Case Study of Prunus persica.

PubMed

Karakülah, Gökhan

2017-06-28

Novel transcript discovery through RNA sequencing has substantially improved our understanding of the transcriptome dynamics of biological systems. Endogenous target mimicry (eTM) transcripts, a novel class of regulatory molecules, bind to their target microRNAs (miRNAs) by base pairing and block their biological activity. The objective of this study was to provide a computational analysis framework for the prediction of putative eTM sequences in plants, and as an example, to discover previously un-annotated eTMs in Prunus persica (peach) transcriptome. Therefore, two public peach transcriptome libraries downloaded from Sequence Read Archive (SRA) and a previously published set of long non-coding RNAs (lncRNAs) were investigated with multi-step analysis pipeline, and 44 putative eTMs were found. Additionally, an eTM-miRNA-mRNA regulatory network module associated with peach fruit organ development was built via integration of the miRNA target information and predicted eTM-miRNA interactions. My findings suggest that one of the most widely expressed miRNA families among diverse plant species, miR156, might be potentially sponged by seven putative eTMs. Besides, the study indicates eTMs potentially play roles in the regulation of development processes in peach fruit via targeting specific miRNAs. In conclusion, by following the step-by step instructions provided in this study, novel eTMs can be identified and annotated effectively in public plant transcriptome libraries.

Harnessing NGS and Big Data Optimally: Comparison of miRNA Prediction from Assembled versus Non-assembled Sequencing Data--The Case of the Grass Aegilops tauschii Complex Genome.

PubMed

Budak, Hikmet; Kantar, Melda

2015-07-01

MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. As high-throughput next generation sequencing (NGS) and Big Data rapidly accumulate for various species, efforts for in silico identification of miRNAs intensify. Surprisingly, the effect of the input genomics sequence on the robustness of miRNA prediction was not evaluated in detail to date. In the present study, we performed a homology-based miRNA and isomiRNA prediction of the 5D chromosome of bread wheat progenitor, Aegilops tauschii, using two distinct sequence data sets as input: (1) raw sequence reads obtained from 454-GS FLX Titanium sequencing platform and (2) an assembly constructed from these reads. We also compared this method with a number of available plant sequence datasets. We report here the identification of 62 and 22 miRNAs from raw reads and the assembly, respectively, of which 16 were predicted with high confidence from both datasets. While raw reads promoted sensitivity with the high number of miRNAs predicted, 55% (12 out of 22) of the assembly-based predictions were supported by previous observations, bringing specificity forward compared to the read-based predictions, of which only 37% were supported. Importantly, raw reads could identify several repeat-related miRNAs that could not be detected with the assembly. However, raw reads could not capture 6 miRNAs, for which the stem-loops could only be covered by the relatively longer sequences from the assembly. In summary, the comparison of miRNA datasets obtained by these two strategies revealed that utilization of raw reads, as well as assemblies for in silico prediction, have distinct advantages and disadvantages. Consideration of these important nuances can benefit future miRNA identification efforts in the current age of NGS and Big Data driven life sciences innovation.

SeqTrim: a high-throughput pipeline for pre-processing any type of sequence read

PubMed Central

2010-01-01

Background High-throughput automated sequencing has enabled an exponential growth rate of sequencing data. This requires increasing sequence quality and reliability in order to avoid database contamination with artefactual sequences. The arrival of pyrosequencing enhances this problem and necessitates customisable pre-processing algorithms. Results SeqTrim has been implemented both as a Web and as a standalone command line application. Already-published and newly-designed algorithms have been included to identify sequence inserts, to remove low quality, vector, adaptor, low complexity and contaminant sequences, and to detect chimeric reads. The availability of several input and output formats allows its inclusion in sequence processing workflows. Due to its specific algorithms, SeqTrim outperforms other pre-processors implemented as Web services or standalone applications. It performs equally well with sequences from EST libraries, SSH libraries, genomic DNA libraries and pyrosequencing reads and does not lead to over-trimming. Conclusions SeqTrim is an efficient pipeline designed for pre-processing of any type of sequence read, including next-generation sequencing. It is easily configurable and provides a friendly interface that allows users to know what happened with sequences at every pre-processing stage, and to verify pre-processing of an individual sequence if desired. The recommended pipeline reveals more information about each sequence than previously described pre-processors and can discard more sequencing or experimental artefacts. PMID:20089148

Inference of Markovian properties of molecular sequences from NGS data and applications to comparative genomics.

PubMed

Ren, Jie; Song, Kai; Deng, Minghua; Reinert, Gesine; Cannon, Charles H; Sun, Fengzhu

2016-04-01

Next-generation sequencing (NGS) technologies generate large amounts of short read data for many different organisms. The fact that NGS reads are generally short makes it challenging to assemble the reads and reconstruct the original genome sequence. For clustering genomes using such NGS data, word-count based alignment-free sequence comparison is a promising approach, but for this approach, the underlying expected word counts are essential.A plausible model for this underlying distribution of word counts is given through modeling the DNA sequence as a Markov chain (MC). For single long sequences, efficient statistics are available to estimate the order of MCs and the transition probability matrix for the sequences. As NGS data do not provide a single long sequence, inference methods on Markovian properties of sequences based on single long sequences cannot be directly used for NGS short read data. Here we derive a normal approximation for such word counts. We also show that the traditional Chi-square statistic has an approximate gamma distribution ,: using the Lander-Waterman model for physical mapping. We propose several methods to estimate the order of the MC based on NGS reads and evaluate those using simulations. We illustrate the applications of our results by clustering genomic sequences of several vertebrate and tree species based on NGS reads using alignment-free sequence dissimilarity measures. We find that the estimated order of the MC has a considerable effect on the clustering results ,: and that the clustering results that use a N: MC of the estimated order give a plausible clustering of the species. Our implementation of the statistics developed here is available as R package 'NGS.MC' at http://www-rcf.usc.edu/∼fsun/Programs/NGS-MC/NGS-MC.html fsun@usc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Improved Annotation of 3′ Untranslated Regions and Complex Loci by Combination of Strand-Specific Direct RNA Sequencing, RNA-Seq and ESTs

PubMed Central

Song, Junfang; Duc, Céline; Storey, Kate G.; McLean, W. H. Irwin; Brown, Sara J.; Simpson, Gordon G.; Barton, Geoffrey J.

2014-01-01

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data. PMID:24722185

Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes.

PubMed

Roy, Subhas Chandra; Moitra, Kaushik; De Sarker, Dilip

2017-01-01

Genetic diversity was assessed in the four orchid species using NGS based ddRAD sequencing data. The assembled nucleotide sequences (fastq) were deposited in the SRA archive of NCBI Database with accession number (SRP063543 for Dendrobium , SRP065790 for Geodorum, SRP072201 for Cymbidium and SRP072378 for Rhynchostylis ). Total base pair read was 1.1 Mbp in case of Dendrobium sp., 553.3 Kbp for Geodorum sp., 1.6 Gbp for Cymbidium , and 1.4 Gbp for Rhynchostylis . Average GC% was 43.9 in Geodorum , 43.7% in Dendrobium , 41.2% in Cymbidium and 42.3% in Rhynchostylis . Four partial gene sequences were used in DnaSP5 program for nucleotide diversity and phylogenetic relationship determination ( Ycf2 gene of Dendrobium, matK gene of Geodorum , psbD gene of Cymbidium and Ycf2 gene of Ryhnchostylis ). Nucleotide diversity (per site) Pi (π) was 0.10560 in Dendrobium, 0.03586 in Geodorum, 0.01364 in Cymbidium and 0.011344 in Rhynchostylis . Neutrality test statistics showed the negative value in all the four orchid species (Tajima's D value -2.17959 in Dendrobium , -2.01655 in Geodorum, -2.12362 in Rhynchostylis and -1.54222 in Cymbidium ) indicating the purifying selection. Result for these gene sequences ( mat K and Ycf 2 and psb D) indicate that they were not evolved neutrally, but signifying that selection might have played a role in evolution of these genes in these four groups of orchids. Phylogenetic relationship was analyzed by reconstructing dendrogram based on the matK, psbD and Ycf2 gene sequences using maximum likelihood method in MEGA6 program.

Draft sequencing and assembly of the genome of the world's largest fish, the whale shark: Rhincodon typus Smith 1828.

PubMed

Read, Timothy D; Petit, Robert A; Joseph, Sandeep J; Alam, Md Tauqeer; Weil, M Ryan; Ahmad, Maida; Bhimani, Ravila; Vuong, Jocelyn S; Haase, Chad P; Webb, D Harry; Tan, Milton; Dove, Alistair D M

2017-07-14

The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

CLAST: CUDA implemented large-scale alignment search tool.

PubMed

Yano, Masahiro; Mori, Hiroshi; Akiyama, Yutaka; Yamada, Takuji; Kurokawa, Ken

2014-12-11

Metagenomics is a powerful methodology to study microbial communities, but it is highly dependent on nucleotide sequence similarity searching against sequence databases. Metagenomic analyses with next-generation sequencing technologies produce enormous numbers of reads from microbial communities, and many reads are derived from microbes whose genomes have not yet been sequenced, limiting the usefulness of existing sequence similarity search tools. Therefore, there is a clear need for a sequence similarity search tool that can rapidly detect weak similarity in large datasets. We developed a tool, which we named CLAST (CUDA implemented large-scale alignment search tool), that enables analyses of millions of reads and thousands of reference genome sequences, and runs on NVIDIA Fermi architecture graphics processing units. CLAST has four main advantages over existing alignment tools. First, CLAST was capable of identifying sequence similarities ~80.8 times faster than BLAST and 9.6 times faster than BLAT. Second, CLAST executes global alignment as the default (local alignment is also an option), enabling CLAST to assign reads to taxonomic and functional groups based on evolutionarily distant nucleotide sequences with high accuracy. Third, CLAST does not need a preprocessed sequence database like Burrows-Wheeler Transform-based tools, and this enables CLAST to incorporate large, frequently updated sequence databases. Fourth, CLAST requires <2 GB of main memory, making it possible to run CLAST on a standard desktop computer or server node. CLAST achieved very high speed (similar to the Burrows-Wheeler Transform-based Bowtie 2 for long reads) and sensitivity (equal to BLAST, BLAT, and FR-HIT) without the need for extensive database preprocessing or a specialized computing platform. Our results demonstrate that CLAST has the potential to be one of the most powerful and realistic approaches to analyze the massive amount of sequence data from next-generation sequencing technologies.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach

PubMed Central

Watson, Mick; Minot, Samuel S.; Rivera, Maria C.; Franklin, Rima B.

2017-01-01

Abstract Background: Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities. PMID:28327976

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

PubMed

Brown, Bonnie L; Watson, Mick; Minot, Samuel S; Rivera, Maria C; Franklin, Rima B

2017-03-01

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities. © The Author 2017. Published by Oxford University Press.

ARKS: chromosome-scale scaffolding of human genome drafts with linked read kmers.

PubMed

Coombe, Lauren; Zhang, Jessica; Vandervalk, Benjamin P; Chu, Justin; Jackman, Shaun D; Birol, Inanc; Warren, René L

2018-06-20

The long-range sequencing information captured by linked reads, such as those available from 10× Genomics (10xG), helps resolve genome sequence repeats, and yields accurate and contiguous draft genome assemblies. We introduce ARKS, an alignment-free linked read genome scaffolding methodology that uses linked reads to organize genome assemblies further into contiguous drafts. Our approach departs from other read alignment-dependent linked read scaffolders, including our own (ARCS), and uses a kmer-based mapping approach. The kmer mapping strategy has several advantages over read alignment methods, including better usability and faster processing, as it precludes the need for input sequence formatting and draft sequence assembly indexing. The reliance on kmers instead of read alignments for pairing sequences relaxes the workflow requirements, and drastically reduces the run time. Here, we show how linked reads, when used in conjunction with Hi-C data for scaffolding, improve a draft human genome assembly of PacBio long-read data five-fold (baseline vs. ARKS NG50 = 4.6 vs. 23.1 Mbp, respectively). We also demonstrate how the method provides further improvements of a megabase-scale Supernova human genome assembly (NG50 = 14.74 Mbp vs. 25.94 Mbp before and after ARKS), which itself exclusively uses linked read data for assembly, with an execution speed six to nine times faster than competitive linked read scaffolders (~ 10.5 h compared to 75.7 h, on average). Following ARKS scaffolding of a human genome 10xG Supernova assembly (of cell line NA12878), fewer than 9 scaffolds cover each chromosome, except the largest (chromosome 1, n = 13). ARKS uses a kmer mapping strategy instead of linked read alignments to record and associate the barcode information needed to order and orient draft assembly sequences. The simplified workflow, when compared to that of our initial implementation, ARCS, markedly improves run time performances on experimental human genome datasets. Furthermore, the novel distance estimator in ARKS utilizes barcoding information from linked reads to estimate gap sizes. It accomplishes this by modeling the relationship between known distances of a region within contigs and calculating associated Jaccard indices. ARKS has the potential to provide correct, chromosome-scale genome assemblies, promptly. We expect ARKS to have broad utility in helping refine draft genomes.

Comparison of CNVs in Buffalo with other species

USDA-ARS?s Scientific Manuscript database

Using a read-depth (RD) and a hybrid read-pair, split-read (RAPTR-SV) CNV detection method, we identified over 1425 unique CNVs in 14 Water Buffalo individual compared to the cattle genome sequence. Total variable sequence of the CNV regions (CNVR) from the RD method approached 59 megabases (~ 2% of...

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver

PubMed Central

Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Cornelissen, Marion; Kellam, Paul; Reiss, Peter

2018-01-01

Abstract Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user’s choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver’s constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver. PMID:29876136

Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

PubMed

Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur

2011-04-18

Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

Are special read alignment strategies necessary and cost-effective when handling sequencing reads from patient-derived tumor xenografts?

PubMed

Tso, Kai-Yuen; Lee, Sau Dan; Lo, Kwok-Wai; Yip, Kevin Y

2014-12-23

Patient-derived tumor xenografts in mice are widely used in cancer research and have become important in developing personalized therapies. When these xenografts are subject to DNA sequencing, the samples could contain various amounts of mouse DNA. It has been unclear how the mouse reads would affect data analyses. We conducted comprehensive simulations to compare three alignment strategies at different mutation rates, read lengths, sequencing error rates, human-mouse mixing ratios and sequenced regions. We also sequenced a nasopharyngeal carcinoma xenograft and a cell line to test how the strategies work on real data. We found the "filtering" and "combined reference" strategies performed better than aligning reads directly to human reference in terms of alignment and variant calling accuracies. The combined reference strategy was particularly good at reducing false negative variants calls without significantly increasing the false positive rate. In some scenarios the performance gain of these two special handling strategies was too small for special handling to be cost-effective, but it was found crucial when false non-synonymous SNVs should be minimized, especially in exome sequencing. Our study systematically analyzes the effects of mouse contamination in the sequencing data of human-in-mouse xenografts. Our findings provide information for designing data analysis pipelines for these data.

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

PubMed Central

Lammers, P J; McLaughlin, S; Papin, S; Trujillo-Provencio, C; Ryncarz, A J

1990-01-01

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria. Images PMID:2123860

Evaluation of Methods for de novo Genome assembly from High-throughput Sequencing Reads Reveals Dependencies that Affect the Quality of the Results

USDA-ARS?s Scientific Manuscript database

Recent developments in high-throughput sequencing technology have made low-cost sequencing an attractive approach for many genome analysis tasks. Increasing read lengths, improving quality and the production of increasingly larger numbers of usable sequences per instrument-run continue to make whole...

Simple tools for assembling and searching high-density picolitre pyrophosphate sequence data.

PubMed

Parker, Nicolas J; Parker, Andrew G

2008-04-18

The advent of pyrophosphate sequencing makes large volumes of sequencing data available at a lower cost than previously possible. However, the short read lengths are difficult to assemble and the large dataset is difficult to handle. During the sequencing of a virus from the tsetse fly, Glossina pallidipes, we found the need for tools to search quickly a set of reads for near exact text matches. A set of tools is provided to search a large data set of pyrophosphate sequence reads under a "live" CD version of Linux on a standard PC that can be used by anyone without prior knowledge of Linux and without having to install a Linux setup on the computer. The tools permit short lengths of de novo assembly, checking of existing assembled sequences, selection and display of reads from the data set and gathering counts of sequences in the reads. Demonstrations are given of the use of the tools to help with checking an assembly against the fragment data set; investigating homopolymer lengths, repeat regions and polymorphisms; and resolving inserted bases caused by incomplete chain extension. The additional information contained in a pyrophosphate sequencing data set beyond a basic assembly is difficult to access due to a lack of tools. The set of simple tools presented here would allow anyone with basic computer skills and a standard PC to access this information.

G-CNV: A GPU-Based Tool for Preparing Data to Detect CNVs with Read-Depth Methods.

PubMed

Manconi, Andrea; Manca, Emanuele; Moscatelli, Marco; Gnocchi, Matteo; Orro, Alessandro; Armano, Giuliano; Milanesi, Luciano

2015-01-01

Copy number variations (CNVs) are the most prevalent types of structural variations (SVs) in the human genome and are involved in a wide range of common human diseases. Different computational methods have been devised to detect this type of SVs and to study how they are implicated in human diseases. Recently, computational methods based on high-throughput sequencing (HTS) are increasingly used. The majority of these methods focus on mapping short-read sequences generated from a donor against a reference genome to detect signatures distinctive of CNVs. In particular, read-depth based methods detect CNVs by analyzing genomic regions with significantly different read-depth from the other ones. The pipeline analysis of these methods consists of four main stages: (i) data preparation, (ii) data normalization, (iii) CNV regions identification, and (iv) copy number estimation. However, available tools do not support most of the operations required at the first two stages of this pipeline. Typically, they start the analysis by building the read-depth signal from pre-processed alignments. Therefore, third-party tools must be used to perform most of the preliminary operations required to build the read-depth signal. These data-intensive operations can be efficiently parallelized on graphics processing units (GPUs). In this article, we present G-CNV, a GPU-based tool devised to perform the common operations required at the first two stages of the analysis pipeline. G-CNV is able to filter low-quality read sequences, to mask low-quality nucleotides, to remove adapter sequences, to remove duplicated read sequences, to map the short-reads, to resolve multiple mapping ambiguities, to build the read-depth signal, and to normalize it. G-CNV can be efficiently used as a third-party tool able to prepare data for the subsequent read-depth signal generation and analysis. Moreover, it can also be integrated in CNV detection tools to generate read-depth signals.

Genovo: De Novo Assembly for Metagenomes

NASA Astrophysics Data System (ADS)

Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

Partial bisulfite conversion for unique template sequencing

PubMed Central

Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael

2018-01-01

Abstract We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. PMID:29161423

Indexing a sequence for mapping reads with a single mismatch.

PubMed

Crochemore, Maxime; Langiu, Alessio; Rahman, M Sohel

2014-05-28

Mapping reads against a genome sequence is an interesting and useful problem in computational molecular biology and bioinformatics. In this paper, we focus on the problem of indexing a sequence for mapping reads with a single mismatch. We first focus on a simpler problem where the length of the pattern is given beforehand during the data structure construction. This version of the problem is interesting in its own right in the context of the next generation sequencing. In the sequel, we show how to solve the more general problem. In both cases, our algorithm can construct an efficient data structure in O(n log(1+ε) n) time and space and can answer subsequent queries in O(m log log n + K) time. Here, n is the length of the sequence, m is the length of the read, 0<ε<1 and is the optimal output size.

Predicted secondary structure similarity in the absence of primary amino acid sequence homology: hepatitis B virus open reading frames.

PubMed Central

Schaeffer, E; Sninsky, J J

1984-01-01

Proteins that are related evolutionarily may have diverged at the level of primary amino acid sequence while maintaining similar secondary structures. Computer analysis has been used to compare the open reading frames of the hepatitis B virus to those of the woodchuck hepatitis virus at the level of amino acid sequence, and to predict the relative hydrophilic character and the secondary structure of putative polypeptides. Similarity is seen at the levels of relative hydrophilicity and secondary structure, in the absence of sequence homology. These data reinforce the proposal that these open reading frames encode viral proteins. Computer analysis of this type can be more generally used to establish structural similarities between proteins that do not share obvious sequence homology as well as to assess whether an open reading frame is fortuitous or codes for a protein. PMID:6585835

Effect of read-mapping biases on detecting allele-specific expression from RNA-sequencing data

PubMed Central

Degner, Jacob F.; Marioni, John C.; Pai, Athma A.; Pickrell, Joseph K.; Nkadori, Everlyne; Gilad, Yoav; Pritchard, Jonathan K.

2009-01-01

Motivation: Next-generation sequencing has become an important tool for genome-wide quantification of DNA and RNA. However, a major technical hurdle lies in the need to map short sequence reads back to their correct locations in a reference genome. Here, we investigate the impact of SNP variation on the reliability of read-mapping in the context of detecting allele-specific expression (ASE). Results: We generated 16 million 35 bp reads from mRNA of each of two HapMap Yoruba individuals. When we mapped these reads to the human genome we found that, at heterozygous SNPs, there was a significant bias toward higher mapping rates of the allele in the reference sequence, compared with the alternative allele. Masking known SNP positions in the genome sequence eliminated the reference bias but, surprisingly, did not lead to more reliable results overall. We find that even after masking, ∼5–10% of SNPs still have an inherent bias toward more effective mapping of one allele. Filtering out inherently biased SNPs removes 40% of the top signals of ASE. The remaining SNPs showing ASE are enriched in genes previously known to harbor cis-regulatory variation or known to show uniparental imprinting. Our results have implications for a variety of applications involving detection of alternate alleles from short-read sequence data. Availability: Scripts, written in Perl and R, for simulating short reads, masking SNP variation in a reference genome and analyzing the simulation output are available upon request from JFD. Raw short read data were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18156. Contact: jdegner@uchicago.edu; marioni@uchicago.edu; gilad@uchicago.edu; pritch@uchicago.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19808877

Recombinative Generalization: An Exploratory Study in Musical Reading

PubMed Central

Perez, William Ferreira; de Rose, Julio C

2010-01-01

The present study aimed to extend the findings of recombinative generalization research in alphabetical reading and spelling to the context of musical reading. One participant was taught to respond discriminatively to six two-note sequences, choosing the corresponding notation on the staff in the presence of each sequence. When novel three- and four-note sequences were presented, she selected the corresponding notation. These results suggest the generality of previous research to the context of musical teaching. PMID:22477462

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

PubMed

Fuselli, S; Baptista, R P; Panziera, A; Magi, A; Guglielmi, S; Tonin, R; Benazzo, A; Bauzer, L G; Mazzoni, C J; Bertorelle, G

2018-03-24

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Genetically optimizing weather predictions

NASA Astrophysics Data System (ADS)

Potter, S. B.; Staats, Kai; Romero-Colmenero, Encarni

2016-07-01

humidity, air pressure, wind speed and wind direction) into a database. Built upon this database, we have developed a remarkably simple approach to derive a functional weather predictor. The aim is provide up to the minute local weather predictions in order to e.g. prepare dome environment conditions ready for night time operations or plan, prioritize and update weather dependent observing queues. In order to predict the weather for the next 24 hours, we take the current live weather readings and search the entire archive for similar conditions. Predictions are made against an averaged, subsequent 24 hours of the closest matches for the current readings. We use an Evolutionary Algorithm to optimize our formula through weighted parameters. The accuracy of the predictor is routinely tested and tuned against the full, updated archive to account for seasonal trends and total, climate shifts. The live (updated every 5 minutes) SALT weather predictor can be viewed here: http://www.saao.ac.za/ sbp/suthweather_predict.html

Zseq: An Approach for Preprocessing Next-Generation Sequencing Data.

PubMed

Alkhateeb, Abedalrhman; Rueda, Luis

2017-08-01

Next-generation sequencing technology generates a huge number of reads (short sequences), which contain a vast amount of genomic data. The sequencing process, however, comes with artifacts. Preprocessing of sequences is mandatory for further downstream analysis. We present Zseq, a linear method that identifies the most informative genomic sequences and reduces the number of biased sequences, sequence duplications, and ambiguous nucleotides. Zseq finds the complexity of the sequences by counting the number of unique k-mers in each sequence as its corresponding score and also takes into the account other factors such as ambiguous nucleotides or high GC-content percentage in k-mers. Based on a z-score threshold, Zseq sweeps through the sequences again and filters those with a z-score less than the user-defined threshold. Zseq algorithm is able to provide a better mapping rate; it reduces the number of ambiguous bases significantly in comparison with other methods. Evaluation of the filtered reads has been conducted by aligning the reads and assembling the transcripts using the reference genome as well as de novo assembly. The assembled transcripts show a better discriminative ability to separate cancer and normal samples in comparison with another state-of-the-art method. Moreover, de novo assembled transcripts from the reads filtered by Zseq have longer genomic sequences than other tested methods. Estimating the threshold of the cutoff point is introduced using labeling rules with optimistic results.

Enhancing the detection of barcoded reads in high throughput DNA sequencing data by controlling the false discovery rate.

PubMed

Buschmann, Tilo; Zhang, Rong; Brash, Douglas E; Bystrykh, Leonid V

2014-08-07

DNA barcodes are short unique sequences used to label DNA or RNA-derived samples in multiplexed deep sequencing experiments. During the demultiplexing step, barcodes must be detected and their position identified. In some cases (e.g., with PacBio SMRT), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements. In our analysis, barcode sequences showed high rates of coincidental similarities with the Mus musculus reference DNA. This problem became more acute when the length of the barcode sequence decreased and the number of barcodes in the set increased. The method presented in this paper controls the tail area-based false discovery rate to distinguish between barcoded and unbarcoded reads. This method helps to establish the highest acceptable minimal distance between reads and barcode sequences. In a proof of concept experiment we correctly detected barcodes in 83% of the reads with a precision of 89%. Sensitivity improved to 99% at 99% precision when the adjacent primer sequence was incorporated in the analysis. The analysis was further improved using a paired end strategy. Following an analysis of the data for sequence variants induced in the Atp1a1 gene of C57BL/6 murine melanocytes by ultraviolet light and conferring resistance to ouabain, we found no evidence of cross-contamination of DNA material between samples. Our method offers a proper quantitative treatment of the problem of detecting barcoded reads in a noisy sequencing environment. It is based on the false discovery rate statistics that allows a proper trade-off between sensitivity and precision to be chosen.

Sex Genotyping of Archival Fixed and Immunolabeled Guinea Pig Cochleas.

PubMed

Depreux, Frédéric F; Czech, Lyubov; Whitlon, Donna S

2018-03-26

For decades, outbred guinea pigs (GP) have been used as research models. Various past research studies using guinea pigs used measures that, unknown at the time, may be sex-dependent, but from which today, archival tissues may be all that remain. We aimed to provide a protocol for sex-typing archival guinea pig tissue, whereby past experiments could be re-evaluated for sex effects. No PCR sex-genotyping protocols existed for GP. We found that published sequence of the GP Sry gene differed from that in two separate GP stocks. We used sequences from other species to deduce PCR primers for Sry. After developing a genomic DNA extraction for archival, fixed, decalcified, immunolabeled, guinea pig cochlear half-turns, we used a multiplex assay (Y-specific Sry; X-specific Dystrophin) to assign sex to tissue as old as 3 years. This procedure should allow reevaluation of prior guinea pig studies in various research areas for the effects of sex on experimental outcomes.

Swine transcriptome characterization by combined Iso-Seq and RNA-seq for annotating the emerging long read-based reference genome

USDA-ARS?s Scientific Manuscript database

PacBio long-read sequencing technology is increasingly popular in genome sequence assembly and transcriptome cataloguing. Recently, a new-generation pig reference genome was assembled based on long reads from this technology. To finely annotate this genome assembly, transcriptomes of nine tissues fr...

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

PubMed

Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

2013-01-01

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Cloud computing for genomic data analysis and collaboration.

PubMed

Langmead, Ben; Nellore, Abhinav

2018-04-01

Next-generation sequencing has made major strides in the past decade. Studies based on large sequencing data sets are growing in number, and public archives for raw sequencing data have been doubling in size every 18 months. Leveraging these data requires researchers to use large-scale computational resources. Cloud computing, a model whereby users rent computers and storage from large data centres, is a solution that is gaining traction in genomics research. Here, we describe how cloud computing is used in genomics for research and large-scale collaborations, and argue that its elasticity, reproducibility and privacy features make it ideally suited for the large-scale reanalysis of publicly available archived data, including privacy-protected data.

GapFiller: a de novo assembly approach to fill the gap within paired reads

PubMed Central

2012-01-01

Background Next Generation Sequencing technologies are able to provide high genome coverages at a relatively low cost. However, due to limited reads' length (from 30 bp up to 200 bp), specific bioinformatics problems have become even more difficult to solve. De novo assembly with short reads, for example, is more complicated at least for two reasons: first, the overall amount of "noisy" data to cope with increased and, second, as the reads' length decreases the number of unsolvable repeats grows. Our work's aim is to go at the root of the problem by providing a pre-processing tool capable to produce (in-silico) longer and highly accurate sequences from a collection of Next Generation Sequencing reads. Results In this paper a seed-and-extend local assembler is presented. The kernel algorithm is a loop that, starting from a read used as seed, keeps extending it using heuristics whose main goal is to produce a collection of error-free and longer sequences. In particular, GapFiller carefully detects reliable overlaps and operates clustering similar reads in order to reconstruct the missing part between the two ends of the same insert. Our tool's output has been validated on 24 experiments using both simulated and real paired reads datasets. The output sequences are declared correct when the seed-mate is found. In the experiments performed, GapFiller was able to extend high percentages of the processed seeds and find their mates, with a false positives rate that turned out to be nearly negligible. Conclusions GapFiller, starting from a sufficiently high short reads coverage, is able to produce high coverages of accurate longer sequences (from 300 bp up to 3500 bp). The procedure to perform safe extensions, together with the mate-found check, turned out to be a powerful criterion to guarantee contigs' correctness. GapFiller has further potential, as it could be applied in a number of different scenarios, including the post-processing validation of insertions/deletions detection pipelines, pre-processing routines on datasets for de novo assembly pipelines, or in any hierarchical approach designed to assemble, analyse or validate pools of sequences. PMID:23095524

Assessing High-Resolution Weather Research and Forecasting (WRF) Forecasts Using an Object-Based Diagnostic Evaluation

DTIC Science & Technology

2014-02-01

Operational Model Archive and Distribution System ( NOMADS ). The RTMA product was generated using a 2-D variational method to assimilate point weather...observations and satellite-derived measurements (National Weather Service, 2013). The products were downloaded using the NOMADS General Regularly...of the completed WRF run" read Start_Date echo $Start_Date echo " " echo "Enter 2- digit , zulu, observation hour (HH) for remapping" read oHH

Detecting SNPs and estimating allele frequencies in clonal bacterial populations by sequencing pooled DNA.

PubMed

Holt, Kathryn E; Teo, Yik Y; Li, Heng; Nair, Satheesh; Dougan, Gordon; Wain, John; Parkhill, Julian

2009-08-15

Here, we present a method for estimating the frequencies of SNP alleles present within pooled samples of DNA using high-throughput short-read sequencing. The method was tested on real data from six strains of the highly monomorphic pathogen Salmonella Paratyphi A, sequenced individually and in a pool. A variety of read mapping and quality-weighting procedures were tested to determine the optimal parameters, which afforded > or =80% sensitivity of SNP detection and strong correlation with true SNP frequency at poolwide read depth of 40x, declining only slightly at read depths 20-40x. The method was implemented in Perl and relies on the opensource software Maq for read mapping and SNP calling. The Perl script is freely available from ftp://ftp.sanger.ac.uk/pub/pathogens/pools/.

Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

PubMed

Evans, Teri; Johnson, Andrew D; Loose, Matthew

2018-01-12

Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

Acceleration of short and long DNA read mapping without loss of accuracy using suffix array.

PubMed

Tárraga, Joaquín; Arnau, Vicente; Martínez, Héctor; Moreno, Raul; Cazorla, Diego; Salavert-Torres, José; Blanquer-Espert, Ignacio; Dopazo, Joaquín; Medina, Ignacio

2014-12-01

HPG Aligner applies suffix arrays for DNA read mapping. This implementation produces a highly sensitive and extremely fast mapping of DNA reads that scales up almost linearly with read length. The approach presented here is faster (over 20× for long reads) and more sensitive (over 98% in a wide range of read lengths) than the current state-of-the-art mappers. HPG Aligner is not only an optimal alternative for current sequencers but also the only solution available to cope with longer reads and growing throughputs produced by forthcoming sequencing technologies. https://github.com/opencb/hpg-aligner. © The Author 2014. Published by Oxford University Press.

An Investigation of the Role of Sequencing in Children's Reading Comprehension

ERIC Educational Resources Information Center

Gouldthorp, Bethanie; Katsipis, Lia; Mueller, Cara

2018-01-01

To date, little is known about the high-level language skills and cognitive processes underlying reading comprehension in children. The present study aimed to investigate whether children with high, compared with low, reading comprehension differ in their sequencing skill, which was defined as the ability to identify and recall the temporal order…

Curriculum Sequencing and the Acquisition of Clock-Reading Skills among Chinese and Flemish Children

ERIC Educational Resources Information Center

Burny, Elise; Valcke, Martin; Desoete, Annemie; Van Luit, Johannes E. Hans

2013-01-01

The present study addresses the impact of the curriculum on primary school children's acquisition of clock-reading knowledge from analog and digital clocks. Focusing on Chinese and Flemish children's clock-reading knowledge, the study is about whether the differences in sequencing of learning and instruction opportunities--as defined by the…

Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

PubMed

Schwessinger, Benjamin; Rathjen, John P

2017-01-01

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

Compression of next-generation sequencing reads aided by highly efficient de novo assembly

PubMed Central

Jones, Daniel C.; Ruzzo, Walter L.; Peng, Xinxia

2012-01-01

We present Quip, a lossless compression algorithm for next-generation sequencing data in the FASTQ and SAM/BAM formats. In addition to implementing reference-based compression, we have developed, to our knowledge, the first assembly-based compressor, using a novel de novo assembly algorithm. A probabilistic data structure is used to dramatically reduce the memory required by traditional de Bruijn graph assemblers, allowing millions of reads to be assembled very efficiently. Read sequences are then stored as positions within the assembled contigs. This is combined with statistical compression of read identifiers, quality scores, alignment information and sequences, effectively collapsing very large data sets to <15% of their original size with no loss of information. Availability: Quip is freely available under the 3-clause BSD license from http://cs.washington.edu/homes/dcjones/quip. PMID:22904078

Ultra-deep mutant spectrum profiling: improving sequencing accuracy using overlapping read pairs.

PubMed

Chen-Harris, Haiyin; Borucki, Monica K; Torres, Clinton; Slezak, Tom R; Allen, Jonathan E

2013-02-12

High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (<0.05%). Our rare variant detection strategies have important implications beyond viral evolution and can be applied to any basic and clinical research area that requires the identification of rare mutations.

HybPiper: Extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment1

PubMed Central

Johnson, Matthew G.; Gardner, Elliot M.; Liu, Yang; Medina, Rafael; Goffinet, Bernard; Shaw, A. Jonathan; Zerega, Nyree J. C.; Wickett, Norman J.

2016-01-01

Premise of the study: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high-throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user-friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae). Methods and Results: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus. Conclusions: HybPiper was designed for Linux and Mac OS X and is freely available at https://github.com/mossmatters/HybPiper. PMID:27437175

Evaluating approaches to find exon chains based on long reads.

PubMed

Kuosmanen, Anna; Norri, Tuukka; Mäkinen, Veli

2018-05-01

Transcript prediction can be modeled as a graph problem where exons are modeled as nodes and reads spanning two or more exons are modeled as exon chains. Pacific Biosciences third-generation sequencing technology produces significantly longer reads than earlier second-generation sequencing technologies, which gives valuable information about longer exon chains in a graph. However, with the high error rates of third-generation sequencing, aligning long reads correctly around the splice sites is a challenging task. Incorrect alignments lead to spurious nodes and arcs in the graph, which in turn lead to incorrect transcript predictions. We survey several approaches to find the exon chains corresponding to long reads in a splicing graph, and experimentally study the performance of these methods using simulated data to allow for sensitivity/precision analysis. Our experiments show that short reads from second-generation sequencing can be used to significantly improve exon chain correctness either by error-correcting the long reads before splicing graph creation, or by using them to create a splicing graph on which the long-read alignments are then projected. We also study the memory and time consumption of various modules, and show that accurate exon chains lead to significantly increased transcript prediction accuracy. The simulated data and in-house scripts used for this article are available at http://www.cs.helsinki.fi/group/gsa/exon-chains/exon-chains-bib.tar.bz2.

Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples.

EPA Science Inventory

Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses us...

Partial bisulfite conversion for unique template sequencing.

PubMed

Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael; Levy, Dan

2018-01-25

We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Encouraging upward thinking with planetary imagery: science as motivator and enrichment for the adult basic skills learner

NASA Astrophysics Data System (ADS)

Fisher, C. G.; Fisher, K. E.

2004-12-01

Engaging students in the process of understanding the world around them in a college level remedial reading program presents an unmitigated challenge. Students previously unimpressed with the educational system become more active participants in their classrooms when high resolution prints from Landsat obtained from the NASA-GSFC education outreach center are used to initially motivate them to perceive the context of their surroundings. In the course, imagery from Landsat that clearly show the Compton Community College track is introduced, moving on to show students similar perspectives on Egyptian pyramids and other remote regions. The satellite imagery makes understanding maps intuitive. After linking these observations from space to both the student's own experience and far away places on earth, students are introduced to the Star-date publication [http://Stardate.org]. Students are encouraged to individually follow the phases of the moon, find constellations, visit the College's telescope on nights when the astronomy class makes observations, and look for meteor showers. NASA and JPL sites are then used to teach students to access the web. Students receive instruction in using computers to navigate the web, where they then follow missions in real time, and access archived imagery and written materials. These sources of reading material are particularly valuable because they are written simply, but follow the scientific convention of addressing readers as colleagues. Notably, students have returned after completing the three-course sequence to literacy and commented on the importance to them of having learned about space in the initial course. They have reported on the excitement of teaching their children and others in the community about what they can see by looking up, and indicated their appreciation of receiving posters and handouts obtained at this meeting by displaying them prominently in their homes. Although not a traditional venue for scientific education, the importance of motivating these adult students to develop not only reading skills, but also increased awareness of the world around them gives a clear impetus for including in Compton College's remedial reading sequence the striking imagery available from our space missions. We propose that outreach directed at instructors of courses at this level would result in significant benefit to an underserved population of students, and invite feedback on ways to accomplish this through existing facilities.

Software for Optical Archive and Retrieval (SOAR) user's guide, version 4.2

NASA Technical Reports Server (NTRS)

Davis, Charles

1991-01-01

The optical disk is an emerging technology. Because it is not a magnetic medium, it offers a number of distinct advantages over the established form of storage, advantages that make it extremely attractive. They are as follows: (1) the ability to store much more data within the same space; (2) the random access characteristics of the Write Once Read Many optical disk; (3) a much longer life than that of traditional storage media; and (4) much greater data access rate. Software for Optical Archive and Retrieval (SOAR) user's guide is presented.

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

PubMed

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches.

PubMed

Lin, Hsin-Hung; Liao, Yu-Chieh

2015-01-01

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.

Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231.

PubMed

Baptista, Rodrigo P; Reis-Cunha, Joao Luis; DeBarry, Jeremy D; Chiari, Egler; Kissinger, Jessica C; Bartholomeu, Daniella C; Macedo, Andrea M

2018-02-14

Next-generation sequencing (NGS) methods are low-cost high-throughput technologies that produce thousands to millions of sequence reads. Despite the high number of raw sequence reads, their short length, relative to Sanger, PacBio or Nanopore reads, complicates the assembly of genomic repeats. Many genome tools are available, but the assembly of highly repetitive genome sequences using only NGS short reads remains challenging. Genome assembly of organisms responsible for important neglected diseases such as Trypanosoma cruzi, the aetiological agent of Chagas disease, is known to be challenging because of their repetitive nature. Only three of six recognized discrete typing units (DTUs) of the parasite have their draft genomes published and therefore genome evolution analyses in the taxon are limited. In this study, we developed a computational workflow to assemble highly repetitive genomes via a combination of de novo and reference-based assembly strategies to better overcome the intrinsic limitations of each, based on Illumina reads. The highly repetitive genome of the human-infecting parasite T. cruzi 231 strain was used as a test subject. The combined-assembly approach shown in this study benefits from the reference-based assembly ability to resolve highly repetitive sequences and from the de novo capacity to assemble genome-specific regions, improving the quality of the assembly. The acceptable confidence obtained by analyzing our results showed that our combined approach is an attractive option to assemble highly repetitive genomes with NGS short reads. Phylogenomic analysis including the 231 strain, the first representative of DTU III whose genome was sequenced, was also performed and provides new insights into T. cruzi genome evolution.

Flexible taxonomic assignment of ambiguous sequencing reads

PubMed Central

2011-01-01

Background To characterize the diversity of bacterial populations in metagenomic studies, sequencing reads need to be accurately assigned to taxonomic units in a given reference taxonomy. Reads that cannot be reliably assigned to a unique leaf in the taxonomy (ambiguous reads) are typically assigned to the lowest common ancestor of the set of species that match it. This introduces a potentially severe error in the estimation of bacteria present in the sample due to false positives, since all species in the subtree rooted at the ancestor are implicitly assigned to the read even though many of them may not match it. Results We present a method that maps each read to a node in the taxonomy that minimizes a penalty score while balancing the relevance of precision and recall in the assignment through a parameter q. This mapping can be obtained in time linear in the number of matching sequences, because LCA queries to the reference taxonomy take constant time. When applied to six different metagenomic datasets, our algorithm produces different taxonomic distributions depending on whether coverage or precision is maximized. Including information on the quality of the reads reduces the number of unassigned reads but increases the number of ambiguous reads, stressing the relevance of our method. Finally, two measures of performance are described and results with a set of artificially generated datasets are discussed. Conclusions The assignment strategy of sequencing reads introduced in this paper is a versatile and a quick method to study bacterial communities. The bacterial composition of the analyzed samples can vary significantly depending on how ambiguous reads are assigned depending on the value of the q parameter. Validation of our results in an artificial dataset confirm that a combination of values of q produces the most accurate results. PMID:21211059

VISA--Vector Integration Site Analysis server: a web-based server to rapidly identify retroviral integration sites from next-generation sequencing.

PubMed

Hocum, Jonah D; Battrell, Logan R; Maynard, Ryan; Adair, Jennifer E; Beard, Brian C; Rawlings, David J; Kiem, Hans-Peter; Miller, Daniel G; Trobridge, Grant D

2015-07-07

Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens. We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads. VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.

The long hold: Storing data at the National Archives

NASA Technical Reports Server (NTRS)

Thibodeau, Kenneth

1992-01-01

The National Archives is, in many respects, in a unique position. For example, I find people from other organizations describing an archival medium as one which will last for three to five years. At the National Archives, we deal with the centuries, not years. From our perspective, there is no archival medium for data storage, and we do not expect there will ever be one. Predicting the long-term future of information technology beyond a mere five or ten years approaches the occult arts. But one prediction is probably safe. It is that the technology will continue to change, at least until analysts start talking about the post-information age. If we did have a medium which lasted a hundred years or longer, we probably would not have a device capable of reading it. The issue of obsolescence, as opposed to media stability, is more complex and more costly. It is especially complex at the National Archives because of two other aspects of our peculiar position. The first aspect is that we deal with incoherent data. The second is that we are charged with satisfying unknown and unknowable requirements. A brief overview of these aspects is presented.

Archival Services and Technologies for Scientific Data

NASA Astrophysics Data System (ADS)

Meyer, Jörg; Hardt, Marcus; Streit, Achim; van Wezel, Jos

2014-06-01

After analysis and publication, there is no need to keep experimental data online on spinning disks. For reliability and costs inactive data is moved to tape and put into a data archive. The data archive must provide reliable access for at least ten years following a recommendation of the German Science Foundation (DFG), but many scientific communities wish to keep data available much longer. Data archival is on the one hand purely a bit preservation activity in order to ensure the bits read are the same as those written years before. On the other hand enough information must be archived to be able to use and interpret the content of the data. The latter is depending on many also community specific factors and remains an areas of much debate among archival specialists. The paper describes the current practice of archival and bit preservation in use for different science communities at KIT for which a combination of organizational services and technical tools are required. The special monitoring to detect tape related errors, the software infrastructure in use as well as the service certification are discussed. Plans and developments at KIT also in the context of the Large Scale Data Management and Analysis (LSDMA) project are presented. The technical advantages of the T10 SCSI Stream Commands (SSC-4) and the Linear Tape File System (LTFS) will have a profound impact on future long term archival of large data sets.

ZOOM Lite: next-generation sequencing data mapping and visualization software

PubMed Central

Zhang, Zefeng; Lin, Hao; Ma, Bin

2010-01-01

High-throughput next-generation sequencing technologies pose increasing demands on the efficiency, accuracy and usability of data analysis software. In this article, we present ZOOM Lite, a software for efficient reads mapping and result visualization. With a kernel capable of mapping tens of millions of Illumina or AB SOLiD sequencing reads efficiently and accurately, and an intuitive graphical user interface, ZOOM Lite integrates reads mapping and result visualization into a easy to use pipeline on desktop PC. The software handles both single-end and paired-end reads, and can output both the unique mapping result or the top N mapping results for each read. Additionally, the software takes a variety of input file formats and outputs to several commonly used result formats. The software is freely available at http://bioinfor.com/zoom/lite/. PMID:20530531

RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

PubMed Central

Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; Akiyama, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin; Hazama, Makoto; Nishine, Tsutomu; Harada, Akira; Yamamoto, Rintaro; Matsumoto, Hiroyuki; Sakaguchi, Sumito; Ikegami, Takashi; Kashiwagi, Katsuya; Fujiwake, Syuji; Inoue, Kouji; Togawa, Yoshiyuki; Izawa, Masaki; Ohara, Eiji; Watahiki, Masanori; Yoneda, Yuko; Ishikawa, Tomokazu; Ozawa, Kaori; Tanaka, Takumi; Matsuura, Shuji; Kawai, Jun; Okazaki, Yasushi; Muramatsu, Masami; Inoue, Yorinao; Kira, Akira; Hayashizaki, Yoshihide

2000-01-01

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3′ end and 5′ end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month. PMID:11076861

Flexible Strategy Use by Students Who Learn Much versus Little from Text: Transitions within Think-Aloud Protocols

ERIC Educational Resources Information Center

Cromley, Jennifer G.; Wills, Theodore W.

2016-01-01

Van den Broek's landscape model explicitly posits sequences of moves during reading in real time. Two other models that implicitly describe sequences of processes during reading are tested in the present research. Coded think-aloud data from 24 undergraduate students reading scientific text were analysed with lag-sequential techniques to compare…

One Size Doesn't Fit All - RefEditor: Building Personalized Diploid Reference Genome to Improve Read Mapping and Genotype Calling in Next Generation Sequencing Studies

PubMed Central

Yuan, Shuai; Johnston, H. Richard; Zhang, Guosheng; Li, Yun; Hu, Yi-Juan; Qin, Zhaohui S.

2015-01-01

With rapid decline of the sequencing cost, researchers today rush to embrace whole genome sequencing (WGS), or whole exome sequencing (WES) approach as the next powerful tool for relating genetic variants to human diseases and phenotypes. A fundamental step in analyzing WGS and WES data is mapping short sequencing reads back to the reference genome. This is an important issue because incorrectly mapped reads affect the downstream variant discovery, genotype calling and association analysis. Although many read mapping algorithms have been developed, the majority of them uses the universal reference genome and do not take sequence variants into consideration. Given that genetic variants are ubiquitous, it is highly desirable if they can be factored into the read mapping procedure. In this work, we developed a novel strategy that utilizes genotypes obtained a priori to customize the universal haploid reference genome into a personalized diploid reference genome. The new strategy is implemented in a program named RefEditor. When applying RefEditor to real data, we achieved encouraging improvements in read mapping, variant discovery and genotype calling. Compared to standard approaches, RefEditor can significantly increase genotype calling consistency (from 43% to 61% at 4X coverage; from 82% to 92% at 20X coverage) and reduce Mendelian inconsistency across various sequencing depths. Because many WGS and WES studies are conducted on cohorts that have been genotyped using array-based genotyping platforms previously or concurrently, we believe the proposed strategy will be of high value in practice, which can also be applied to the scenario where multiple NGS experiments are conducted on the same cohort. The RefEditor sources are available at https://github.com/superyuan/refeditor. PMID:26267278

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study.

PubMed

Cerdeira, Louise Teixeira; Carneiro, Adriana Ribeiro; Ramos, Rommel Thiago Jucá; de Almeida, Sintia Silva; D'Afonseca, Vivian; Schneider, Maria Paula Cruz; Baumbach, Jan; Tauch, Andreas; McCulloch, John Anthony; Azevedo, Vasco Ariston Carvalho; Silva, Artur

2011-08-01

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former. Copyright © 2011 Elsevier B.V. All rights reserved.

COPS: A Sensitive and Accurate Tool for Detecting Somatic Copy Number Alterations Using Short-Read Sequence Data from Paired Samples

PubMed Central

Krishnan, Neeraja M.; Gaur, Prakhar; Chaudhary, Rakshit; Rao, Arjun A.; Panda, Binay

2012-01-01

Copy Number Alterations (CNAs) such as deletions and duplications; compose a larger percentage of genetic variations than single nucleotide polymorphisms or other structural variations in cancer genomes that undergo major chromosomal re-arrangements. It is, therefore, imperative to identify cancer-specific somatic copy number alterations (SCNAs), with respect to matched normal tissue, in order to understand their association with the disease. We have devised an accurate, sensitive, and easy-to-use tool, COPS, COpy number using Paired Samples, for detecting SCNAs. We rigorously tested the performance of COPS using short sequence simulated reads at various sizes and coverage of SCNAs, read depths, read lengths and also with real tumor:normal paired samples. We found COPS to perform better in comparison to other known SCNA detection tools for all evaluated parameters, namely, sensitivity (detection of true positives), specificity (detection of false positives) and size accuracy. COPS performed well for sequencing reads of all lengths when used with most upstream read alignment tools. Additionally, by incorporating a downstream boundary segmentation detection tool, the accuracy of SCNA boundaries was further improved. Here, we report an accurate, sensitive and easy to use tool in detecting cancer-specific SCNAs using short-read sequence data. In addition to cancer, COPS can be used for any disease as long as sequence reads from both disease and normal samples from the same individual are available. An added boundary segmentation detection module makes COPS detected SCNA boundaries more specific for the samples studied. COPS is available at ftp://115.119.160.213 with username “cops” and password “cops”. PMID:23110103

SMRT sequencing of the Vitis vinifera cv. ‘Flame seedless’ genome using a SMRTbell-free library preparation from Swift Biosciences

USDA-ARS?s Scientific Manuscript database

Single Molecule Real-Time (SMRT) sequencing provides advantages to the sequencing of complex genomes. The long reads generated are superior for resolving complex genomic regions and provide highly contiguous de novo assemblies. Current SMRTbell libraries generate average read lengths of 10-15kb. How...

Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts

PubMed Central

Cheng, Bing; Furtado, Agnelo

2017-01-01

Abstract Polyploidization contributes to the complexity of gene expression, resulting in numerous related but different transcripts. This study explored the transcriptome diversity and complexity of the tetraploid Arabica coffee (Coffea arabica) bean. Long-read sequencing (LRS) by Pacbio Isoform sequencing (Iso-seq) was used to obtain full-length transcripts without the difficulty and uncertainty of assembly required for reads from short-read technologies. The tetraploid transcriptome was annotated and compared with data from the sub-genome progenitors. Caffeine and sucrose genes were targeted for case analysis. An isoform-level tetraploid coffee bean reference transcriptome with 95 995 distinct transcripts (average 3236 bp) was obtained. A total of 88 715 sequences (92.42%) were annotated with BLASTx against NCBI non-redundant plant proteins, including 34 719 high-quality annotations. Further BLASTn analysis against NCBI non-redundant nucleotide sequences, Coffea canephora coding sequences with UTR, C. arabica ESTs, and Rfam resulted in 1213 sequences without hits, were potential novel genes in coffee. Longer UTRs were captured, especially in the 5΄UTRs, facilitating the identification of upstream open reading frames. The LRS also revealed more and longer transcript variants in key caffeine and sucrose metabolism genes from this polyploid genome. Long sequences (>10 kilo base) were poorly annotated. LRS technology shows the limitation of previous studies. It provides an important tool to produce a reference transcriptome including more of the diversity of full-length transcripts to help understand the biology and support the genetic improvement of polyploid species such as coffee. PMID:29048540

Impact of sequencing depth in ChIP-seq experiments

PubMed Central

Jung, Youngsook L.; Luquette, Lovelace J.; Ho, Joshua W.K.; Ferrari, Francesco; Tolstorukov, Michael; Minoda, Aki; Issner, Robbyn; Epstein, Charles B.; Karpen, Gary H.; Kuroda, Mitzi I.; Park, Peter J.

2014-01-01

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data. PMID:24598259

svviz: a read viewer for validating structural variants.

PubMed

Spies, Noah; Zook, Justin M; Salit, Marc; Sidow, Arend

2015-12-15

Visualizing read alignments is the most effective way to validate candidate structural variants (SVs) with existing data. We present svviz, a sequencing read visualizer for SVs that sorts and displays only reads relevant to a candidate SV. svviz works by searching input bam(s) for potentially relevant reads, realigning them against the inferred sequence of the putative variant allele as well as the reference allele and identifying reads that match one allele better than the other. Separate views of the two alleles are then displayed in a scrollable web browser view, enabling a more intuitive visualization of each allele, compared with the single reference genome-based view common to most current read browsers. The browser view facilitates examining the evidence for or against a putative variant, estimating zygosity, visualizing affected genomic annotations and manual refinement of breakpoints. svviz supports data from most modern sequencing platforms. svviz is implemented in python and freely available from http://svviz.github.io/. Published by Oxford University Press 2015. This work is written by US Government employees and is in the public domain in the US.

Louisa May Alcott's Orchard House

Science.gov Websites

and Her Father! Buy the book from Orchard House Read more about John Matteson's award here Louisa May comments about this site, click to use our Online Guest Book All archival photographs © Louisa May Online Guest Book

Improving de novo sequence assembly using machine learning and comparative genomics for overlap correction.

PubMed

Palmer, Lance E; Dejori, Mathaeus; Bolanos, Randall; Fasulo, Daniel

2010-01-15

With the rapid expansion of DNA sequencing databases, it is now feasible to identify relevant information from prior sequencing projects and completed genomes and apply it to de novo sequencing of new organisms. As an example, this paper demonstrates how such extra information can be used to improve de novo assemblies by augmenting the overlapping step. Finding all pairs of overlapping reads is a key task in many genome assemblers, and to this end, highly efficient algorithms have been developed to find alignments in large collections of sequences. It is well known that due to repeated sequences, many aligned pairs of reads nevertheless do not overlap. But no overlapping algorithm to date takes a rigorous approach to separating aligned but non-overlapping read pairs from true overlaps. We present an approach that extends the Minimus assembler by a data driven step to classify overlaps as true or false prior to contig construction. We trained several different classification models within the Weka framework using various statistics derived from overlaps of reads available from prior sequencing projects. These statistics included percent mismatch and k-mer frequencies within the overlaps as well as a comparative genomics score derived from mapping reads to multiple reference genomes. We show that in real whole-genome sequencing data from the E. coli and S. aureus genomes, by providing a curated set of overlaps to the contigging phase of the assembler, we nearly doubled the median contig length (N50) without sacrificing coverage of the genome or increasing the number of mis-assemblies. Machine learning methods that use comparative and non-comparative features to classify overlaps as true or false can be used to improve the quality of a sequence assembly.

Genome assembly from synthetic long read clouds

PubMed Central

Kuleshov, Volodymyr; Snyder, Michael P.; Batzoglou, Serafim

2016-01-01

Motivation: Despite rapid progress in sequencing technology, assembling de novo the genomes of new species as well as reconstructing complex metagenomes remains major technological challenges. New synthetic long read (SLR) technologies promise significant advances towards these goals; however, their applicability is limited by high sequencing requirements and the inability of current assembly paradigms to cope with combinations of short and long reads. Results: Here, we introduce Architect, a new de novo scaffolder aimed at SLR technologies. Unlike previous assembly strategies, Architect does not require a costly subassembly step; instead it assembles genomes directly from the SLR’s underlying short reads, which we refer to as read clouds. This enables a 4- to 20-fold reduction in sequencing requirements and a 5-fold increase in assembly contiguity on both genomic and metagenomic datasets relative to state-of-the-art assembly strategies aimed directly at fully subassembled long reads. Availability and Implementation: Our source code is freely available at https://github.com/kuleshov/architect. Contact: kuleshov@stanford.edu PMID:27307620

Cyber infrastructure for Fusarium: three integrated platforms supporting strain identification, phylogenetics, comparative genomics and knowledge sharing.

PubMed

Park, Bongsoo; Park, Jongsun; Cheong, Kyeong-Chae; Choi, Jaeyoung; Jung, Kyongyong; Kim, Donghan; Lee, Yong-Hwan; Ward, Todd J; O'Donnell, Kerry; Geiser, David M; Kang, Seogchan

2011-01-01

The fungal genus Fusarium includes many plant and/or animal pathogenic species and produces diverse toxins. Although accurate species identification is critical for managing such threats, it is difficult to identify Fusarium morphologically. Fortunately, extensive molecular phylogenetic studies, founded on well-preserved culture collections, have established a robust foundation for Fusarium classification. Genomes of four Fusarium species have been published with more being currently sequenced. The Cyber infrastructure for Fusarium (CiF; http://www.fusariumdb.org/) was built to support archiving and utilization of rapidly increasing data and knowledge and consists of Fusarium-ID, Fusarium Comparative Genomics Platform (FCGP) and Fusarium Community Platform (FCP). The Fusarium-ID archives phylogenetic marker sequences from most known species along with information associated with characterized isolates and supports strain identification and phylogenetic analyses. The FCGP currently archives five genomes from four species. Besides supporting genome browsing and analysis, the FCGP presents computed characteristics of multiple gene families and functional groups. The Cart/Favorite function allows users to collect sequences from Fusarium-ID and the FCGP and analyze them later using multiple tools without requiring repeated copying-and-pasting of sequences. The FCP is designed to serve as an online community forum for sharing and preserving accumulated experience and knowledge to support future research and education.

Cyber infrastructure for Fusarium: three integrated platforms supporting strain identification, phylogenetics, comparative genomics and knowledge sharing

PubMed Central

Park, Bongsoo; Park, Jongsun; Cheong, Kyeong-Chae; Choi, Jaeyoung; Jung, Kyongyong; Kim, Donghan; Lee, Yong-Hwan; Ward, Todd J.; O'Donnell, Kerry; Geiser, David M.; Kang, Seogchan

2011-01-01

The fungal genus Fusarium includes many plant and/or animal pathogenic species and produces diverse toxins. Although accurate species identification is critical for managing such threats, it is difficult to identify Fusarium morphologically. Fortunately, extensive molecular phylogenetic studies, founded on well-preserved culture collections, have established a robust foundation for Fusarium classification. Genomes of four Fusarium species have been published with more being currently sequenced. The Cyber infrastructure for Fusarium (CiF; http://www.fusariumdb.org/) was built to support archiving and utilization of rapidly increasing data and knowledge and consists of Fusarium-ID, Fusarium Comparative Genomics Platform (FCGP) and Fusarium Community Platform (FCP). The Fusarium-ID archives phylogenetic marker sequences from most known species along with information associated with characterized isolates and supports strain identification and phylogenetic analyses. The FCGP currently archives five genomes from four species. Besides supporting genome browsing and analysis, the FCGP presents computed characteristics of multiple gene families and functional groups. The Cart/Favorite function allows users to collect sequences from Fusarium-ID and the FCGP and analyze them later using multiple tools without requiring repeated copying-and-pasting of sequences. The FCP is designed to serve as an online community forum for sharing and preserving accumulated experience and knowledge to support future research and education. PMID:21087991

Minimap2: pairwise alignment for nucleotide sequences.

PubMed

Li, Heng

2018-05-10

Recent advances in sequencing technologies promise ultra-long reads of ∼100 kilo bases (kb) in average, full-length mRNA or cDNA reads in high throughput and genomic contigs over 100 mega bases (Mb) in length. Existing alignment programs are unable or inefficient to process such data at scale, which presses for the development of new alignment algorithms. Minimap2 is a general-purpose alignment program to map DNA or long mRNA sequences against a large reference database. It works with accurate short reads of ≥ 100bp in length, ≥1kb genomic reads at error rate ∼15%, full-length noisy Direct RNA or cDNA reads, and assembly contigs or closely related full chromosomes of hundreds of megabases in length. Minimap2 does split-read alignment, employs concave gap cost for long insertions and deletions (INDELs) and introduces new heuristics to reduce spurious alignments. It is 3-4 times as fast as mainstream short-read mappers at comparable accuracy, and is ≥30 times faster than long-read genomic or cDNA mappers at higher accuracy, surpassing most aligners specialized in one type of alignment. https://github.com/lh3/minimap2. hengli@broadinstitute.org.

BBMerge – Accurate paired shotgun read merging via overlap

DOE PAGES

Bushnell, Brian; Rood, Jonathan; Singer, Esther

2017-10-26

Merging paired-end shotgun reads generated on high-throughput sequencing platforms can substantially improve various subsequent bioinformatics processes, including genome assembly, binning, mapping, annotation, and clustering for taxonomic analysis. With the inexorable growth of sequence data volume and CPU core counts, the speed and scalability of read-processing tools becomes ever-more important. The accuracy of shotgun read merging is crucial as well, as errors introduced by incorrect merging percolate through to reduce the quality of downstream analysis. Thus, we designed a new tool to maximize accuracy and minimize processing time, allowing the use of read merging on larger datasets, and in analyses highlymore » sensitive to errors. We present BBMerge, a new merging tool for paired-end shotgun sequence data. We benchmark BBMerge by comparison with eight other widely used merging tools, assessing speed, accuracy and scalability. Evaluations of both synthetic and real-world datasets demonstrate that BBMerge produces merged shotgun reads with greater accuracy and at higher speed than any existing merging tool examined. BBMerge also provides the ability to merge non-overlapping shotgun read pairs by using k-mer frequency information to assemble the unsequenced gap between reads, achieving a significantly higher merge rate while maintaining or increasing accuracy.« less

BBMerge – Accurate paired shotgun read merging via overlap

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bushnell, Brian; Rood, Jonathan; Singer, Esther

Merging paired-end shotgun reads generated on high-throughput sequencing platforms can substantially improve various subsequent bioinformatics processes, including genome assembly, binning, mapping, annotation, and clustering for taxonomic analysis. With the inexorable growth of sequence data volume and CPU core counts, the speed and scalability of read-processing tools becomes ever-more important. The accuracy of shotgun read merging is crucial as well, as errors introduced by incorrect merging percolate through to reduce the quality of downstream analysis. Thus, we designed a new tool to maximize accuracy and minimize processing time, allowing the use of read merging on larger datasets, and in analyses highlymore » sensitive to errors. We present BBMerge, a new merging tool for paired-end shotgun sequence data. We benchmark BBMerge by comparison with eight other widely used merging tools, assessing speed, accuracy and scalability. Evaluations of both synthetic and real-world datasets demonstrate that BBMerge produces merged shotgun reads with greater accuracy and at higher speed than any existing merging tool examined. BBMerge also provides the ability to merge non-overlapping shotgun read pairs by using k-mer frequency information to assemble the unsequenced gap between reads, achieving a significantly higher merge rate while maintaining or increasing accuracy.« less

Genotype calling from next-generation sequencing data using haplotype information of reads

PubMed Central

Zhi, Degui; Wu, Jihua; Liu, Nianjun; Zhang, Kui

2012-01-01

Motivation: Low coverage sequencing provides an economic strategy for whole genome sequencing. When sequencing a set of individuals, genotype calling can be challenging due to low sequencing coverage. Linkage disequilibrium (LD) based refinement of genotyping calling is essential to improve the accuracy. Current LD-based methods use read counts or genotype likelihoods at individual potential polymorphic sites (PPSs). Reads that span multiple PPSs (jumping reads) can provide additional haplotype information overlooked by current methods. Results: In this article, we introduce a new Hidden Markov Model (HMM)-based method that can take into account jumping reads information across adjacent PPSs and implement it in the HapSeq program. Our method extends the HMM in Thunder and explicitly models jumping reads information as emission probabilities conditional on the states of adjacent PPSs. Our simulation results show that, compared to Thunder, HapSeq reduces the genotyping error rate by 30%, from 0.86% to 0.60%. The results from the 1000 Genomes Project show that HapSeq reduces the genotyping error rate by 12 and 9%, from 2.24% and 2.76% to 1.97% and 2.50% for individuals with European and African ancestry, respectively. We expect our program can improve genotyping qualities of the large number of ongoing and planned whole genome sequencing projects. Contact: dzhi@ms.soph.uab.edu; kzhang@ms.soph.uab.edu Availability: The software package HapSeq and its manual can be found and downloaded at www.ssg.uab.edu/hapseq/. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22285565

DNA Sequencing Using capillary Electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dr. Barry Karger

2011-05-09

The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linkedmore » polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other application papers of sequencing up to this level were also published in the mid 1990's. A major interest of the sequencing community has always been read length. The longer the sequence read per run the more efficient the process as well as the ability to read repeat sequences. We therefore devoted a great deal of time to studying the factors influencing read length in capillary electrophoresis, including polymer type and molecule weight, capillary column temperature, applied electric field, etc. In our initial optimization, we were able to demonstrate, for the first time, the sequencing of over 1000 bases with 90% accuracy. The run required 80 minutes for separation. Sequencing of 1000 bases per column was next demonstrated on a multiple capillary instrument. Our studies revealed that linear polyacrylamide produced the longest read lengths because the hydrophilic single strand DNA had minimal interaction with the very hydrophilic linear polyacrylamide. Any interaction of the DNA with the polymer would lead to broader peaks and lower read length. Another important parameter was the molecular weight of the linear chains. High molecular weight (> 1 MDA) was important to allow the long single strand DNA to reptate through the entangled polymer matrix. In an important paper, we showed an inverse emulsion method to prepare reproducibility linear polyacrylamide polymer with an average MWT of 9MDa. This approach was used in the polymer for sequencing the human genome. Another critical factor in the successful use of capillary electrophoresis for sequencing was the sample preparation method. In the Sanger sequencing reaction, high concentration of salts and dideoxynucleotide remained. Since the sample was introduced to the capillary column by electrokinetic injection, these salt ions would be favorably injected into the column over the sequencing fragments, thus reducing the signal for longer fragments and hence reading read length. In two papers, we examined the role of individual components from the sequencing reaction and then developed a protocol to reduce the deleterious salts. We demonstrated a robust method for achieving long read length DNA sequencing. Continuing our advances, we next demonstrated the achievement of over 1000 bases in less than one hour with a base calling accuracy of between 98 and 99%. In this work, we implemented energy transfer dyes which allowed for cleaner differentiation of the 4 dye labeled terminal nucleotides. In addition, we developed improved base calling software to help read sequencing when the separation was only minimal as occurs at long read lengths. Another critical parameter we studied was column temperature. We demonstrated that read lengths improved as the column temperature was increased from room temperature to 60 C or 70 C. The higher temperature relaxed the DNA chains under the influence of the high electric field.« less

DistMap: a toolkit for distributed short read mapping on a Hadoop cluster.

PubMed

Pandey, Ram Vinay; Schlötterer, Christian

2013-01-01

With the rapid and steady increase of next generation sequencing data output, the mapping of short reads has become a major data analysis bottleneck. On a single computer, it can take several days to map the vast quantity of reads produced from a single Illumina HiSeq lane. In an attempt to ameliorate this bottleneck we present a new tool, DistMap - a modular, scalable and integrated workflow to map reads in the Hadoop distributed computing framework. DistMap is easy to use, currently supports nine different short read mapping tools and can be run on all Unix-based operating systems. It accepts reads in FASTQ format as input and provides mapped reads in a SAM/BAM format. DistMap supports both paired-end and single-end reads thereby allowing the mapping of read data produced by different sequencing platforms. DistMap is available from http://code.google.com/p/distmap/

DistMap: A Toolkit for Distributed Short Read Mapping on a Hadoop Cluster

PubMed Central

Pandey, Ram Vinay; Schlötterer, Christian

2013-01-01

With the rapid and steady increase of next generation sequencing data output, the mapping of short reads has become a major data analysis bottleneck. On a single computer, it can take several days to map the vast quantity of reads produced from a single Illumina HiSeq lane. In an attempt to ameliorate this bottleneck we present a new tool, DistMap - a modular, scalable and integrated workflow to map reads in the Hadoop distributed computing framework. DistMap is easy to use, currently supports nine different short read mapping tools and can be run on all Unix-based operating systems. It accepts reads in FASTQ format as input and provides mapped reads in a SAM/BAM format. DistMap supports both paired-end and single-end reads thereby allowing the mapping of read data produced by different sequencing platforms. DistMap is available from http://code.google.com/p/distmap/ PMID:24009693

MBMC: An Effective Markov Chain Approach for Binning Metagenomic Reads from Environmental Shotgun Sequencing Projects.

PubMed

Wang, Ying; Hu, Haiyan; Li, Xiaoman

2016-08-01

Metagenomics is a next-generation omics field currently impacting postgenomic life sciences and medicine. Binning metagenomic reads is essential for the understanding of microbial function, compositions, and interactions in given environments. Despite the existence of dozens of computational methods for metagenomic read binning, it is still very challenging to bin reads. This is especially true for reads from unknown species, from species with similar abundance, and/or from low-abundance species in environmental samples. In this study, we developed a novel taxonomy-dependent and alignment-free approach called MBMC (Metagenomic Binning by Markov Chains). Different from all existing methods, MBMC bins reads by measuring the similarity of reads to the trained Markov chains for different taxa instead of directly comparing reads with known genomic sequences. By testing on more than 24 simulated and experimental datasets with species of similar abundance, species of low abundance, and/or unknown species, we report here that MBMC reliably grouped reads from different species into separate bins. Compared with four existing approaches, we demonstrated that the performance of MBMC was comparable with existing approaches when binning reads from sequenced species, and superior to existing approaches when binning reads from unknown species. MBMC is a pivotal tool for binning metagenomic reads in the current era of Big Data and postgenomic integrative biology. The MBMC software can be freely downloaded at http://hulab.ucf.edu/research/projects/metagenomics/MBMC.html .

DOE final report 100608.doc

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golden, Susan S

2008-10-16

The aim of this project was to inactivate each locus of the genome of the cyanobacterium Synechococcus elongatus PCC 7942 and screen resulting mutants for altered circadian phenotypes. The immediate goal was to identify all open reading frames (ORFs) that contribute to circadian timing. An additional result was to create a complete archived set of mutagenesis templates, of great utility for the wider research community, that will allow inactivation of any given locus in the genome of S. elongatus. Clones that carry segments of the S. elongatus genome were saturated with transposon insertions in vitro. We completed saturation mutagenesis ofmore » the chromosome (~2800 ORFs). The positions of insertions were sequenced for 17,767 mutagenized clones. Each individual insertion into the S. elongatus DNA in a cosmid or plasmid is a substrate for mutagenesis of the genome via homologous recombination. Because the complete insertion mutation clone set is 5-7 fold redundant, we produced a streamlined set of clones that contains one insertion mutation per locus in the genome, a unigene set. All clones are archived as Escherichia coli stocks frozen in glycerol in 96-well plates at -85ºC and as replicas of these plates on Whatman CloneSaver cards. Each of the mutagenesis substrates from the unigene set has been recombined into the chromosome of wild-type S. elongatus and these cyanobacterial mutants have been archived at -85ºC as well. S. elongatus insertion mutants defective for than 1400 independent genes have screened in luciferase reporter gene backgrounds to evaluate the effect of each mutation on circadian rhythms of gene expression. For the first 700 genes tested, mutagenesis of 71 different ORFs resulted in circadian phenotypes. The mutagenesis project also created insertion mutations in the endogenous large plasmid of S. elongatus, pANL. The sequence of pANL revealed two potential addiction cassettes that appear to account for selection for plasmid persistence. Genetic experiments confirmed that these regions are present on all sub-sets of the plasmid that can replace wild-type pANL. Analysis of mutants defective in each of the remaining ~1400 genes for defects in circadian rhythms will be completed with support from another agency as part of a larger project on circadian rhythms in this cyanobacterium.« less

Evaluation of sampling and storage procedures on preserving the community structure of stool microbiota: A simple at-home toilet-paper collection method.

PubMed

Al, Kait F; Bisanz, Jordan E; Gloor, Gregory B; Reid, Gregor; Burton, Jeremy P

2018-01-01

The increasing interest on the impact of the gut microbiota on health and disease has resulted in multiple human microbiome-related studies emerging. However, multiple sampling methods are being used, making cross-comparison of results difficult. To avoid additional clinic visits and increase patient recruitment to these studies, there is the potential to utilize at-home stool sampling. The aim of this pilot study was to compare simple self-sampling collection and storage methods. To simulate storage conditions, stool samples from three volunteers were freshly collected, placed on toilet tissue, and stored at four temperatures (-80, 7, 22 and 37°C), either dry or in the presence of a stabilization agent (RNAlater®) for 3 or 7days. Using 16S rRNA gene sequencing by Illumina, the effect of storage variations for each sample was compared to a reference community from fresh, unstored counterparts. Fastq files may be accessed in the NCBI Sequence Read Archive: Bioproject ID PRJNA418287. Microbial diversity and composition were not significantly altered by any storage method. Samples were always separable based on participant, regardless of storage method suggesting there was no need for sample preservation by a stabilization agent. In summary, if immediate sample processing is not feasible, short term storage of unpreserved stool samples on toilet paper offers a reliable way to assess the microbiota composition by 16S rRNA gene sequencing. Copyright © 2017 Elsevier B.V. All rights reserved.

ARYANA: Aligning Reads by Yet Another Approach

PubMed Central

2014-01-01

Motivation Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $106 prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. Contribution We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. Availability ARYANA with complete source code can be obtained from http://github.com/aryana-aligner PMID:25252881

ARYANA: Aligning Reads by Yet Another Approach.

PubMed

Gholami, Milad; Arbabi, Aryan; Sharifi-Zarchi, Ali; Chitsaz, Hamidreza; Sadeghi, Mehdi

2014-01-01

Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $10(6) prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. ARYANA with complete source code can be obtained from http://github.com/aryana-aligner.

Automated Finishing with Autofinish

PubMed Central

Gordon, David; Desmarais, Cindy; Green, Phil

2001-01-01

Currently, the genome sequencing community is producing shotgun sequence data at a very high rate, but finishing (collecting additional directed sequence data to close gaps and improve the quality of the data) is not matching that rate. One reason for the difference is that shotgun sequencing is highly automated but finishing is not: Most finishing decisions, such as which directed reads to obtain and which specialized sequencing techniques to use, are made by people. If finishing rates are to increase to match shotgun sequencing rates, most finishing decisions also must be automated. The Autofinish computer program (which is part of the Consed computer software package) does this by automatically choosing finishing reads. Autofinish is able to suggest most finishing reads required for completion of each sequencing project, greatly reducing the amount of human attention needed. Autofinish sometimes completely finishes the project, with no human decisions required. It cannot solve the most complex problems, so we recommend that Autofinish be allowed to suggest reads for the first three rounds of finishing, and if the project still is not finished completely, a human finisher complete the work. We compared this Autofinish-Hybrid method of finishing against a human finisher in five different projects with a variety of shotgun depths by finishing each project twice—once with each method. This comparison shows that the Autofinish-Hybrid method saves many hours over a human finisher alone, while using roughly the same number and type of reads and closing gaps at roughly the same rate. Autofinish currently is in production use at several large sequencing centers. It is designed to be adaptable to the finishing strategy of the lab—it can finish using some or all of the following: resequencing reads, reverses, custom primer walks on either subclone templates or whole clone templates, PCR, or minilibraries. Autofinish has been used for finishing cDNA, genomic clones, and whole bacterial genomes (see http://www.phrap.org). PMID:11282977

Teaching Reading and the At Risk Pupil.

ERIC Educational Resources Information Center

Ediger, Marlow

At risk students need to experience a reading curriculum which offers success in learning to read; appropriate sequence of reading activities; feedback regarding what has been accomplished in reading; rewards for doing well when comparing past with present achievement records; intrinsic motivation in wanting to read; help and guidance to achieve…

Bloomfield-Barnhart Developmental Reading Mastery Test: Evaluator's Handbook [and] Individual Pupil Packet.

ERIC Educational Resources Information Center

Barnhart, Cynthia A.; And Others

The Developmental Reading Mastery Test of oral reading, comprehension, spelling, and language skills is based on curriculum materials by the Bloomfield-Barnhart reading program, Let's Read. The non-graded program teaches reading in eleven steps (skill sequences), with corresponding subtests, as follows: (1) shape discrimination, directionality;…

Application of long sequence reads to improve genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7

DOE PAGES

Utturkar, Sagar M.; Bayer, Edward A.; Borovok, Ilya; ...

2016-09-29

Here, we and others have shown the utility of long sequence reads to improve genome assembly quality. In this study, we generated PacBio DNA sequence data to improve the assemblies of draft genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7.

Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors.

PubMed

Paasinen-Sohns, Aino; Koelzer, Viktor H; Frank, Angela; Schafroth, Julian; Gisler, Aline; Sachs, Melanie; Graber, Anne; Rothschild, Sacha I; Wicki, Andreas; Cathomas, Gieri; Mertz, Kirsten D

2017-03-01

Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of sequencing depth on the characterization of the microbiome and resistome.

PubMed

Zaheer, Rahat; Noyes, Noelle; Ortega Polo, Rodrigo; Cook, Shaun R; Marinier, Eric; Van Domselaar, Gary; Belk, Keith E; Morley, Paul S; McAllister, Tim A

2018-04-12

Developments in high-throughput next generation sequencing (NGS) technology have rapidly advanced the understanding of overall microbial ecology as well as occurrence and diversity of specific genes within diverse environments. In the present study, we compared the ability of varying sequencing depths to generate meaningful information about the taxonomic structure and prevalence of antimicrobial resistance genes (ARGs) in the bovine fecal microbial community. Metagenomic sequencing was conducted on eight composite fecal samples originating from four beef cattle feedlots. Metagenomic DNA was sequenced to various depths, D1, D0.5 and D0.25, with average sample read counts of 117, 59 and 26 million, respectively. A comparative analysis of the relative abundance of reads aligning to different phyla and antimicrobial classes indicated that the relative proportions of read assignments remained fairly constant regardless of depth. However, the number of reads being assigned to ARGs as well as to microbial taxa increased significantly with increasing depth. We found a depth of D0.5 was suitable to describe the microbiome and resistome of cattle fecal samples. This study helps define a balance between cost and required sequencing depth to acquire meaningful results.

Study design requirements for RNA sequencing-based breast cancer diagnostics.

PubMed

Mer, Arvind Singh; Klevebring, Daniel; Grönberg, Henrik; Rantalainen, Mattias

2016-02-01

Sequencing-based molecular characterization of tumors provides information required for individualized cancer treatment. There are well-defined molecular subtypes of breast cancer that provide improved prognostication compared to routine biomarkers. However, molecular subtyping is not yet implemented in routine breast cancer care. Clinical translation is dependent on subtype prediction models providing high sensitivity and specificity. In this study we evaluate sample size and RNA-sequencing read requirements for breast cancer subtyping to facilitate rational design of translational studies. We applied subsampling to ascertain the effect of training sample size and the number of RNA sequencing reads on classification accuracy of molecular subtype and routine biomarker prediction models (unsupervised and supervised). Subtype classification accuracy improved with increasing sample size up to N = 750 (accuracy = 0.93), although with a modest improvement beyond N = 350 (accuracy = 0.92). Prediction of routine biomarkers achieved accuracy of 0.94 (ER) and 0.92 (Her2) at N = 200. Subtype classification improved with RNA-sequencing library size up to 5 million reads. Development of molecular subtyping models for cancer diagnostics requires well-designed studies. Sample size and the number of RNA sequencing reads directly influence accuracy of molecular subtyping. Results in this study provide key information for rational design of translational studies aiming to bring sequencing-based diagnostics to the clinic.

Technical Report on Modeling for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLoughlin, K.

2016-01-11

The overall aim of this project is to develop a software package, called MetaQuant, that can determine the constituents of a complex microbial sample and estimate their relative abundances by analysis of metagenomic sequencing data. The goal for Task 1 is to create a generative model describing the stochastic process underlying the creation of sequence read pairs in the data set. The stages in this generative process include the selection of a source genome sequence for each read pair, with probability dependent on its abundance in the sample. The other stages describe the evolution of the source genome from itsmore » nearest common ancestor with a reference genome, breakage of the source DNA into short fragments, and the errors in sequencing the ends of the fragments to produce read pairs.« less

Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data.

PubMed

Rask, Thomas S; Petersen, Bent; Chen, Donald S; Day, Karen P; Pedersen, Anders Gorm

2016-04-22

Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data. The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data. This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon sequencing data, particularly where extensive prior knowledge is available about the obtained sequences, for example in analysis of the immunoglobulin VDJ region where Multipass can be combined with a model for the known recombining germline genes. Multipass is available for Roche 454 data at http://www.cbs.dtu.dk/services/MultiPass-1.0 , and the concept can potentially be implemented for other sequencing technologies as well.

RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

PubMed Central

Munger, Steven C.; Raghupathy, Narayanan; Choi, Kwangbom; Simons, Allen K.; Gatti, Daniel M.; Hinerfeld, Douglas A.; Svenson, Karen L.; Keller, Mark P.; Attie, Alan D.; Hibbs, Matthew A.; Graber, Joel H.; Chesler, Elissa J.; Churchill, Gary A.

2014-01-01

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations. PMID:25236449

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

PubMed

Chen, He; Yao, Jiacheng; Fu, Yusi; Pang, Yuhong; Wang, Jianbin; Huang, Yanyi

2018-04-11

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

PubMed Central

Tsai, Yu-Chih; Deming, Clayton; Segre, Julia A.; Kong, Heidi H.; Korlach, Jonas

2016-01-01

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. PMID:26861018

An accurate algorithm for the detection of DNA fragments from dilution pool sequencing experiments.

PubMed

Bansal, Vikas

2018-01-01

The short read lengths of current high-throughput sequencing technologies limit the ability to recover long-range haplotype information. Dilution pool methods for preparing DNA sequencing libraries from high molecular weight DNA fragments enable the recovery of long DNA fragments from short sequence reads. These approaches require computational methods for identifying the DNA fragments using aligned sequence reads and assembling the fragments into long haplotypes. Although a number of computational methods have been developed for haplotype assembly, the problem of identifying DNA fragments from dilution pool sequence data has not received much attention. We formulate the problem of detecting DNA fragments from dilution pool sequencing experiments as a genome segmentation problem and develop an algorithm that uses dynamic programming to optimize a likelihood function derived from a generative model for the sequence reads. This algorithm uses an iterative approach to automatically infer the mean background read depth and the number of fragments in each pool. Using simulated data, we demonstrate that our method, FragmentCut, has 25-30% greater sensitivity compared with an HMM based method for fragment detection and can also detect overlapping fragments. On a whole-genome human fosmid pool dataset, the haplotypes assembled using the fragments identified by FragmentCut had greater N50 length, 16.2% lower switch error rate and 35.8% lower mismatch error rate compared with two existing methods. We further demonstrate the greater accuracy of our method using two additional dilution pool datasets. FragmentCut is available from https://bansal-lab.github.io/software/FragmentCut. vibansal@ucsd.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing. PMID:24236095

A filtering method to generate high quality short reads using illumina paired-end technology.

PubMed

Eren, A Murat; Vineis, Joseph H; Morrison, Hilary G; Sogin, Mitchell L

2013-01-01

Consensus between independent reads improves the accuracy of genome and transcriptome analyses, however lack of consensus between very similar sequences in metagenomic studies can and often does represent natural variation of biological significance. The common use of machine-assigned quality scores on next generation platforms does not necessarily correlate with accuracy. Here, we describe using the overlap of paired-end, short sequence reads to identify error-prone reads in marker gene analyses and their contribution to spurious OTUs following clustering analysis using QIIME. Our approach can also reduce error in shotgun sequencing data generated from libraries with small, tightly constrained insert sizes. The open-source implementation of this algorithm in Python programming language with user instructions can be obtained from https://github.com/meren/illumina-utils.

Open Reading Frame Phylogenetic Analysis on the Cloud

PubMed Central

2013-01-01

Phylogenetic analysis has become essential in researching the evolutionary relationships between viruses. These relationships are depicted on phylogenetic trees, in which viruses are grouped based on sequence similarity. Viral evolutionary relationships are identified from open reading frames rather than from complete sequences. Recently, cloud computing has become popular for developing internet-based bioinformatics tools. Biocloud is an efficient, scalable, and robust bioinformatics computing service. In this paper, we propose a cloud-based open reading frame phylogenetic analysis service. The proposed service integrates the Hadoop framework, virtualization technology, and phylogenetic analysis methods to provide a high-availability, large-scale bioservice. In a case study, we analyze the phylogenetic relationships among Norovirus. Evolutionary relationships are elucidated by aligning different open reading frame sequences. The proposed platform correctly identifies the evolutionary relationships between members of Norovirus. PMID:23671843

Detecting and Estimating Contamination of Human DNA Samples in Sequencing and Array-Based Genotype Data

PubMed Central

Jun, Goo; Flickinger, Matthew; Hetrick, Kurt N.; Romm, Jane M.; Doheny, Kimberly F.; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

2012-01-01

DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies. PMID:23103226

Identification of genomic indels and structural variations using split reads

PubMed Central

2011-01-01

Background Recent studies have demonstrated the genetic significance of insertions, deletions, and other more complex structural variants (SVs) in the human population. With the development of the next-generation sequencing technologies, high-throughput surveys of SVs on the whole-genome level have become possible. Here we present split-read identification, calibrated (SRiC), a sequence-based method for SV detection. Results We start by mapping each read to the reference genome in standard fashion using gapped alignment. Then to identify SVs, we score each of the many initial mappings with an assessment strategy designed to take into account both sequencing and alignment errors (e.g. scoring more highly events gapped in the center of a read). All current SV calling methods have multilevel biases in their identifications due to both experimental and computational limitations (e.g. calling more deletions than insertions). A key aspect of our approach is that we calibrate all our calls against synthetic data sets generated from simulations of high-throughput sequencing (with realistic error models). This allows us to calculate sensitivity and the positive predictive value under different parameter-value scenarios and for different classes of events (e.g. long deletions vs. short insertions). We run our calculations on representative data from the 1000 Genomes Project. Coupling the observed numbers of events on chromosome 1 with the calibrations gleaned from the simulations (for different length events) allows us to construct a relatively unbiased estimate for the total number of SVs in the human genome across a wide range of length scales. We estimate in particular that an individual genome contains ~670,000 indels/SVs. Conclusions Compared with the existing read-depth and read-pair approaches for SV identification, our method can pinpoint the exact breakpoints of SV events, reveal the actual sequence content of insertions, and cover the whole size spectrum for deletions. Moreover, with the advent of the third-generation sequencing technologies that produce longer reads, we expect our method to be even more useful. PMID:21787423

De novo assembly and phasing of a Korean human genome.

PubMed

Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

2016-10-13

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

75 FR 16516 - Dates Correction

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-01

... NATIONAL ARCHIVES AND RECORDS ADMINISTRATION Office of the Federal Register Dates Correction Correction In the Notices section beginning on page 15401 in the issue of March 29th, 2010, make the following correction: On pages 15401 through 15499, the date at the top of each page is corrected to read...

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly

PubMed Central

2013-01-01

Background The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. Results We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. Conclusions These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies. PMID:23496952

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly.

PubMed

Francis, Warren R; Christianson, Lynne M; Kiko, Rainer; Powers, Meghan L; Shaner, Nathan C; Haddock, Steven H D

2013-03-12

The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies.

MetaMap: An atlas of metatranscriptomic reads in human disease-related RNA-seq data.

PubMed

Simon, L M; Karg, S; Westermann, A J; Engel, M; Elbehery, A H A; Hense, B; Heinig, M; Deng, L; Theis, F J

2018-06-12

With the advent of the age of big data in bioinformatics, large volumes of data and high performance computing power enable researchers to perform re-analyses of publicly available datasets at an unprecedented scale. Ever more studies imply the microbiome in both normal human physiology and a wide range of diseases. RNA sequencing technology (RNA-seq) is commonly used to infer global eukaryotic gene expression patterns under defined conditions, including human disease-related contexts, but its generic nature also enables the detection of microbial and viral transcripts. We developed a bioinformatic pipeline to screen existing human RNA-seq datasets for the presence of microbial and viral reads by re-inspecting the non-human-mapping read fraction. We validated this approach by recapitulating outcomes from 6 independent controlled infection experiments of cell line models and comparison with an alternative metatranscriptomic mapping strategy. We then applied the pipeline to close to 150 terabytes of publicly available raw RNA-seq data from >17,000 samples from >400 studies relevant to human disease using state-of-the-art high performance computing systems. The resulting data of this large-scale re-analysis are made available in the presented MetaMap resource. Our results demonstrate that common human RNA-seq data, including those archived in public repositories, might contain valuable information to correlate microbial and viral detection patterns with diverse diseases. The presented MetaMap database thus provides a rich resource for hypothesis generation towards the role of the microbiome in human disease. Additionally, codes to process new datasets and perform statistical analyses are made available at https://github.com/theislab/MetaMap.

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

PubMed

Jayakumar, Vasanthan; Sakakibara, Yasubumi

2017-11-03

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

Patient information: confidentiality and the electronic record.

PubMed

Griffith, Richard

The rise of the electronic record now allows nurses to access a large archive of patient information that was more difficult to obtain when records consisted of manually held paper files. There have been several instances where curiosity and, occasionally, more malicious motivations have led nurses to access these records and read the notes of a celebrity or a person they know. In this article, Richard Griffith considers whether nurses' accessing and reading of the record of someone who is not in their care is in breach of their duty of confidentiality.

Fiscal and Operational Impacts of Standardizing US Military Resiliency Programs to Minimize Post-Traumatic Stress Disorder

DTIC Science & Technology

2016-04-01

the Vietnam conflict, and 15 years prior to the introduction of the concept of PTSD, Herbert Archibald and Read Tuddenham were examining patients...Psychiatric Pub- lishing, 2013. Archibald, Herbert C., and Read D. Tuddenham. “Persistent Stress Reac- tion after Combat: A 20-Year Follow-Up.” Archives of...mean-less-stigma /2012/05/05/gIQAlV8M4T_story.html. 34 Johnson, Spencer . Who Moved My Cheese? An Amazing Way to Deal with Change in Your Work and in

Site handbook: data acquisition system information, Shelly Ridge Program Center for the Girl Scouts of greater Philadelphia, Miquon, Pennsylvania

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1985-01-01

Data were collected at the Shelly Ridge Girl Scout Center using an Aeolian Kinetics PDL-24 data acquisition system. Instantaneous data readings were recorded each 15 seconds by the microprocessor. These channel readings were then averaged to produce hourly values which were then stored on an audio cassette. These hourly data were then transcribed to the AIAF archive. The Girl Scout Center features an 861 square foot unvented Trombe wall, a direct gain sunspace, and two rooftop collectors for heating domestic water.

Comparison of mapping algorithms used in high-throughput sequencing: application to Ion Torrent data

PubMed Central

2014-01-01

Background The rapid evolution in high-throughput sequencing (HTS) technologies has opened up new perspectives in several research fields and led to the production of large volumes of sequence data. A fundamental step in HTS data analysis is the mapping of reads onto reference sequences. Choosing a suitable mapper for a given technology and a given application is a subtle task because of the difficulty of evaluating mapping algorithms. Results In this paper, we present a benchmark procedure to compare mapping algorithms used in HTS using both real and simulated datasets and considering four evaluation criteria: computational resource and time requirements, robustness of mapping, ability to report positions for reads in repetitive regions, and ability to retrieve true genetic variation positions. To measure robustness, we introduced a new definition for a correctly mapped read taking into account not only the expected start position of the read but also the end position and the number of indels and substitutions. We developed CuReSim, a new read simulator, that is able to generate customized benchmark data for any kind of HTS technology by adjusting parameters to the error types. CuReSim and CuReSimEval, a tool to evaluate the mapping quality of the CuReSim simulated reads, are freely available. We applied our benchmark procedure to evaluate 14 mappers in the context of whole genome sequencing of small genomes with Ion Torrent data for which such a comparison has not yet been established. Conclusions A benchmark procedure to compare HTS data mappers is introduced with a new definition for the mapping correctness as well as tools to generate simulated reads and evaluate mapping quality. The application of this procedure to Ion Torrent data from the whole genome sequencing of small genomes has allowed us to validate our benchmark procedure and demonstrate that it is helpful for selecting a mapper based on the intended application, questions to be addressed, and the technology used. This benchmark procedure can be used to evaluate existing or in-development mappers as well as to optimize parameters of a chosen mapper for any application and any sequencing platform. PMID:24708189

On missing Data Treatment for degraded video and film archives: a survey and a new Bayesian approach.

PubMed

Kokaram, Anil C

2004-03-01

Image sequence restoration has been steadily gaining in importance with the increasing prevalence of visual digital media. The demand for content increases the pressure on archives to automate their restoration activities for preservation of the cultural heritage that they hold. There are many defects that affect archived visual material and one central issue is that of Dirt and Sparkle, or "Blotches." Research in archive restoration has been conducted for more than a decade and this paper places that material in context to highlight the advances made during that time. The paper also presents a new and simpler Bayesian framework that achieves joint processing of noise, missing data, and occlusion.

RSEQtools: a modular framework to analyze RNA-Seq data using compact, anonymized data summaries.

PubMed

Habegger, Lukas; Sboner, Andrea; Gianoulis, Tara A; Rozowsky, Joel; Agarwal, Ashish; Snyder, Michael; Gerstein, Mark

2011-01-15

The advent of next-generation sequencing for functional genomics has given rise to quantities of sequence information that are often so large that they are difficult to handle. Moreover, sequence reads from a specific individual can contain sufficient information to potentially identify and genetically characterize that person, raising privacy concerns. In order to address these issues, we have developed the Mapped Read Format (MRF), a compact data summary format for both short and long read alignments that enables the anonymization of confidential sequence information, while allowing one to still carry out many functional genomics studies. We have developed a suite of tools (RSEQtools) that use this format for the analysis of RNA-Seq experiments. These tools consist of a set of modules that perform common tasks such as calculating gene expression values, generating signal tracks of mapped reads and segmenting that signal into actively transcribed regions. Moreover, the tools can readily be used to build customizable RNA-Seq workflows. In addition to the anonymization afforded by MRF, this format also facilitates the decoupling of the alignment of reads from downstream analyses. RSEQtools is implemented in C and the source code is available at http://rseqtools.gersteinlab.org/.

Aligner optimization increases accuracy and decreases compute times in multi-species sequence data.

PubMed

Robinson, Kelly M; Hawkins, Aziah S; Santana-Cruz, Ivette; Adkins, Ricky S; Shetty, Amol C; Nagaraj, Sushma; Sadzewicz, Lisa; Tallon, Luke J; Rasko, David A; Fraser, Claire M; Mahurkar, Anup; Silva, Joana C; Dunning Hotopp, Julie C

2017-09-01

As sequencing technologies have evolved, the tools to analyze these sequences have made similar advances. However, for multi-species samples, we observed important and adverse differences in alignment specificity and computation time for bwa- mem (Burrows-Wheeler aligner-maximum exact matches) relative to bwa-aln. Therefore, we sought to optimize bwa-mem for alignment of data from multi-species samples in order to reduce alignment time and increase the specificity of alignments. In the multi-species cases examined, there was one majority member (i.e. Plasmodium falciparum or Brugia malayi ) and one minority member (i.e. human or the Wolbachia endosymbiont w Bm) of the sequence data. Increasing bwa-mem seed length from the default value reduced the number of read pairs from the majority sequence member that incorrectly aligned to the reference genome of the minority sequence member. Combining both source genomes into a single reference genome increased the specificity of mapping, while also reducing the central processing unit (CPU) time. In Plasmodium , at a seed length of 18 nt, 24.1 % of reads mapped to the human genome using 1.7±0.1 CPU hours, while 83.6 % of reads mapped to the Plasmodium genome using 0.2±0.0 CPU hours (total: 107.7 % reads mapping; in 1.9±0.1 CPU hours). In contrast, 97.1 % of the reads mapped to a combined Plasmodium- human reference in only 0.7±0.0 CPU hours. Overall, the results suggest that combining all references into a single reference database and using a 23 nt seed length reduces the computational time, while maximizing specificity. Similar results were found for simulated sequence reads from a mock metagenomic data set. We found similar improvements to computation time in a publicly available human-only data set.

Repeat-aware modeling and correction of short read errors.

PubMed

Yang, Xiao; Aluru, Srinivas; Dorman, Karin S

2011-02-15

High-throughput short read sequencing is revolutionizing genomics and systems biology research by enabling cost-effective deep coverage sequencing of genomes and transcriptomes. Error detection and correction are crucial to many short read sequencing applications including de novo genome sequencing, genome resequencing, and digital gene expression analysis. Short read error detection is typically carried out by counting the observed frequencies of kmers in reads and validating those with frequencies exceeding a threshold. In case of genomes with high repeat content, an erroneous kmer may be frequently observed if it has few nucleotide differences with valid kmers with multiple occurrences in the genome. Error detection and correction were mostly applied to genomes with low repeat content and this remains a challenging problem for genomes with high repeat content. We develop a statistical model and a computational method for error detection and correction in the presence of genomic repeats. We propose a method to infer genomic frequencies of kmers from their observed frequencies by analyzing the misread relationships among observed kmers. We also propose a method to estimate the threshold useful for validating kmers whose estimated genomic frequency exceeds the threshold. We demonstrate that superior error detection is achieved using these methods. Furthermore, we break away from the common assumption of uniformly distributed errors within a read, and provide a framework to model position-dependent error occurrence frequencies common to many short read platforms. Lastly, we achieve better error correction in genomes with high repeat content. The software is implemented in C++ and is freely available under GNU GPL3 license and Boost Software V1.0 license at "http://aluru-sun.ece.iastate.edu/doku.php?id = redeem". We introduce a statistical framework to model sequencing errors in next-generation reads, which led to promising results in detecting and correcting errors for genomes with high repeat content.

Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing.

PubMed

Hong, Jungeui; Gresham, David

2017-11-01

Quantitative analysis of next-generation sequencing (NGS) data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are identified as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. Misidentification of these molecules as PCR duplicates can introduce unforeseen biases during analyses. Here, we developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed, single-index sequencing. Incorporation of UMIs into TruSeq adapters (TrUMIseq adapters) enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TrUMIseq adapters, we show that accurate removal of PCR duplicates results in improved accuracy of both allele frequency (AF) estimation in heterogeneous populations using DNA sequencing and gene expression quantification using RNA-Seq.

miRBase: integrating microRNA annotation and deep-sequencing data.

PubMed

Kozomara, Ana; Griffiths-Jones, Sam

2011-01-01

miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/.

Sequence Data for Clostridium autoethanogenum using Three Generations of Sequencing Technologies

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn Marie; Bruno-Barcena, José M.; ...

2015-04-14

During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20 kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequencemore » datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data.« less

Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome.

PubMed

Saha, Surya; Hunter, Wayne B; Reese, Justin; Morgan, J Kent; Marutani-Hert, Mizuri; Huang, Hong; Lindeberg, Magdalen

2012-01-01

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.

Survey of Endosymbionts in the Diaphorina citri Metagenome and Assembly of a Wolbachia wDi Draft Genome

PubMed Central

Saha, Surya; Hunter, Wayne B.; Reese, Justin; Morgan, J. Kent; Marutani-Hert, Mizuri; Huang, Hong; Lindeberg, Magdalen

2012-01-01

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China. PMID:23166822

An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-01-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Author 2017. Published by Oxford University Press.

Erratum to: An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-10-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Authors 2017. Published by Oxford University Press.

10 CFR 2.206 - Requests for action under this subpart.

Code of Federal Regulations, 2011 CFR

2011-01-01

... manner that enables the NRC to receive, read, authenticate, distribute, and archive the submission, and... [email protected]; or by writing the Office of Information Services, U.S. Nuclear Regulatory... authorized to extend the time for Commission review on its own motion of a Director's denial under paragraph...

10 CFR 2.206 - Requests for action under this subpart.

Code of Federal Regulations, 2010 CFR

2010-01-01

... manner that enables the NRC to receive, read, authenticate, distribute, and archive the submission, and... [email protected]; or by writing the Office of Information Services, U.S. Nuclear Regulatory... authorized to extend the time for Commission review on its own motion of a Director's denial under paragraph...

Composing Change: The Role of Graduate Education in Sustaining a Digital Scholarly Future

ERIC Educational Resources Information Center

Blair, Kristine L.

2014-01-01

In "Reading the Archives: Ten Years on Nonlinear ("Kairos") History," James Kalmbach acknowledges the significant role graduate students have played as digital innovators in the field, particularly in the formation of "Kairos: A Journal of Rhetoric, Technology, Pedagogy" in 1996. Graduate students in the Rhetoric and…

Esprit de Place: Maintaining and Designing Library Buildings To Provide Transcendent Spaces.

ERIC Educational Resources Information Center

Demas, Sam; Scherer, Jeffrey A.

2002-01-01

Discusses library buildings and their role in building community. Reviews current design trends, including reading and study spaces; collaborative workspaces; technology-free zones; archives and special collections; cultural events spaces; age-specific spaces; shared spaces; natural light and landscapes; and interior design trends. (LRW)

Macro and Microenvironments at the British Library.

ERIC Educational Resources Information Center

Shenton, Helen

This paper describes the storage of the 12 million items that have just been moved into the new British Library building. The specifications for the storage and environmental conditions for different types of library and archive material are explained. The varying environmental parameters for storage areas and public areas, including reading rooms…

BarraCUDA - a fast short read sequence aligner using graphics processing units

PubMed Central

2012-01-01

Background With the maturation of next-generation DNA sequencing (NGS) technologies, the throughput of DNA sequencing reads has soared to over 600 gigabases from a single instrument run. General purpose computing on graphics processing units (GPGPU), extracts the computing power from hundreds of parallel stream processors within graphics processing cores and provides a cost-effective and energy efficient alternative to traditional high-performance computing (HPC) clusters. In this article, we describe the implementation of BarraCUDA, a GPGPU sequence alignment software that is based on BWA, to accelerate the alignment of sequencing reads generated by these instruments to a reference DNA sequence. Findings Using the NVIDIA Compute Unified Device Architecture (CUDA) software development environment, we ported the most computational-intensive alignment component of BWA to GPU to take advantage of the massive parallelism. As a result, BarraCUDA offers a magnitude of performance boost in alignment throughput when compared to a CPU core while delivering the same level of alignment fidelity. The software is also capable of supporting multiple CUDA devices in parallel to further accelerate the alignment throughput. Conclusions BarraCUDA is designed to take advantage of the parallelism of GPU to accelerate the alignment of millions of sequencing reads generated by NGS instruments. By doing this, we could, at least in part streamline the current bioinformatics pipeline such that the wider scientific community could benefit from the sequencing technology. BarraCUDA is currently available from http://seqbarracuda.sf.net PMID:22244497

STORMSeq: an open-source, user-friendly pipeline for processing personal genomics data in the cloud.

PubMed

Karczewski, Konrad J; Fernald, Guy Haskin; Martin, Alicia R; Snyder, Michael; Tatonetti, Nicholas P; Dudley, Joel T

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5-10 hours to process a full exome sequence and $30 and 3-8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2.

The Genome Sequencer FLX System--longer reads, more applications, straight forward bioinformatics and more complete data sets.

PubMed

Droege, Marcus; Hill, Brendon

2008-08-31

The Genome Sequencer FLX System (GS FLX), powered by 454 Sequencing, is a next-generation DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and ultra-high throughput. It has been proven to be the most versatile of all currently available next-generation sequencing technologies, supporting many high-profile studies in over seven applications categories. GS FLX users have pursued innovative research in de novo sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics, and RNA analysis. 454 Sequencing is a powerful tool for human genetics research, having recently re-sequenced the genome of an individual human, currently re-sequencing the complete human exome and targeted genomic regions using the NimbleGen sequence capture process, and detected low-frequency somatic mutations linked to cancer.

Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads

PubMed Central

Mourier, Tobias; Mollerup, Sarah; Vinner, Lasse; Hansen, Thomas Arn; Kjartansdóttir, Kristín Rós; Guldberg Frøslev, Tobias; Snogdal Boutrup, Torsten; Nielsen, Lars Peter; Willerslev, Eske; Hansen, Anders J.

2015-01-01

From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses. PMID:26493184

Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation.

PubMed

Hara, Yuichiro; Tatsumi, Kaori; Yoshida, Michio; Kajikawa, Eriko; Kiyonari, Hiroshi; Kuraku, Shigehiro

2015-11-18

RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity. Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs. To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities. Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600more » reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: Anauthor video summaryof this article is available.« less

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

PubMed Central

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin

2016-01-01

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available. PMID:27822530

IM-TORNADO: a tool for comparison of 16S reads from paired-end libraries.

PubMed

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.

PubMed

Haiminen, Niina; Feltus, F Alex; Parida, Laxmi

2011-04-15

We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.

Fast imputation using medium- or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Direct imputation from raw sequence reads can be more accurate than calling genotypes first and then imputing, especially if read depth is low or error rates high, but different imputation strategies are required than those used for data from genotyping chips. A fast algorithm to impute from lower t...

Derivational Suffixes as Cues to Stress Position in Reading Greek

ERIC Educational Resources Information Center

Grimani, Aikaterini; Protopapas, Athanassios

2017-01-01

Background: In languages with lexical stress, reading aloud must include stress assignment. Stress information sources across languages include word-final letter sequences. Here, we examine whether such sequences account for stress assignment in Greek and whether this is attributable to absolute rules involving accenting morphemes or to…

Assembled sequence contigs by SOAPdenova and Volvet algorithms from metagenomic short reads of a new bacterial isolate of gut origin

USDA-ARS?s Scientific Manuscript database

Assembled sequence contigs by SOAPdenova and Volvet algorithms from metagenomic short reads of a new bacterial isolate of gut origin. This study included 2 submissions with a total of 9.8 million bp of assembled contigs....

Additional annotation of the pig transcriptome using integrated Iso-seq and Illumina RNA-seq analysis

USDA-ARS?s Scientific Manuscript database

Alternative splicing is a well-known phenomenon that dramatically increases eukaryotic transcriptome diversity. The extent of mRNA isoform diversity among porcine tissues was assessed using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) and Illumina short read sequencing ...

The use of museum specimens with high-throughput DNA sequencers

PubMed Central

Burrell, Andrew S.; Disotell, Todd R.; Bergey, Christina M.

2015-01-01

Natural history collections have long been used by morphologists, anatomists, and taxonomists to probe the evolutionary process and describe biological diversity. These biological archives also offer great opportunities for genetic research in taxonomy, conservation, systematics, and population biology. They allow assays of past populations, including those of extinct species, giving context to present patterns of genetic variation and direct measures of evolutionary processes. Despite this potential, museum specimens are difficult to work with because natural postmortem processes and preservation methods fragment and damage DNA. These problems have restricted geneticists’ ability to use natural history collections primarily by limiting how much of the genome can be surveyed. Recent advances in DNA sequencing technology, however, have radically changed this, making truly genomic studies from museum specimens possible. We review the opportunities and drawbacks of the use of museum specimens, and suggest how to best execute projects when incorporating such samples. Several high-throughput (HT) sequencing methodologies, including whole genome shotgun sequencing, sequence capture, and restriction digests (demonstrated here), can be used with archived biomaterials. PMID:25532801

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

PubMed Central

Nowrousian, Minou; Stajich, Jason E.; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D.; Pöggeler, Stefanie; Read, Nick D.; Seiler, Stephan; Smith, Kristina M.; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-01-01

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology. PMID:20386741

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

PubMed

Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Reading on the Internet: Realizing and Constructing Potential Texts

ERIC Educational Resources Information Center

Cho, Byeong-Young; Afflerbach, Peter

2015-01-01

Successful Internet reading requires making strategic decisions about what texts to read and a sequence for reading them, all in accordance with readers' goals. In this paper, we describe the process of realizing and constructing potential texts as an important and critical part of successful Internet reading and use verbal report data to…

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

PubMed Central

2011-01-01

Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed. PMID:21794110

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes.

PubMed

Feltus, Frank A; Saski, Christopher A; Mockaitis, Keithanne; Haiminen, Niina; Parida, Laxmi; Smith, Zachary; Ford, James; Staton, Margaret E; Ficklin, Stephen P; Blackmon, Barbara P; Cheng, Chun-Huai; Schnell, Raymond J; Kuhn, David N; Motamayor, Juan-Carlos

2011-07-27

BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Deep sequencing of evolving pathogen populations: applications, errors, and bioinformatic solutions

PubMed Central

2014-01-01

Deep sequencing harnesses the high throughput nature of next generation sequencing technologies to generate population samples, treating information contained in individual reads as meaningful. Here, we review applications of deep sequencing to pathogen evolution. Pioneering deep sequencing studies from the virology literature are discussed, such as whole genome Roche-454 sequencing analyses of the dynamics of the rapidly mutating pathogens hepatitis C virus and HIV. Extension of the deep sequencing approach to bacterial populations is then discussed, including the impacts of emerging sequencing technologies. While it is clear that deep sequencing has unprecedented potential for assessing the genetic structure and evolutionary history of pathogen populations, bioinformatic challenges remain. We summarise current approaches to overcoming these challenges, in particular methods for detecting low frequency variants in the context of sequencing error and reconstructing individual haplotypes from short reads. PMID:24428920

Restoration of Apollo Data by the Lunar Data Project/PDS Lunar Data Node: An Update

NASA Technical Reports Server (NTRS)

Williams, David R.; Hills, H. Kent; Taylor, Patrick T.; Grayzeck, Edwin J.; Guinness, Edward A.

2016-01-01

The Apollo 11, 12, and 14 through 17 missions orbited and landed on the Moon, carrying scientific instruments that returned data from all phases of the missions, included long-lived Apollo Lunar Surface Experiments Packages (ALSEPs) deployed by the astronauts on the lunar surface. Much of these data were never archived, and some of the archived data were on media and in formats that are outmoded, or were deposited with little or no useful documentation to aid outside users. This is particularly true of the ALSEP data returned autonomously for many years after the Apollo missions ended. The purpose of the Lunar Data Project and the Planetary Data System (PDS) Lunar Data Node is to take data collections already archived at the NASA Space Science Data Coordinated Archive (NSSDCA) and prepare them for archiving through PDS, and to locate lunar data that were never archived, bring them into NSSDCA, and then archive them through PDS. Preparing these data for archiving involves reading the data from the original media, be it magnetic tape, microfilm, microfiche, or hard-copy document, converting the outmoded, often binary, formats when necessary, putting them into a standard digital form accepted by PDS, collecting the necessary ancillary data and documentation (metadata) to ensure that the data are usable and well-described, summarizing the metadata in documentation to be included in the data set, adding other information such as references, mission and instrument descriptions, contact information, and related documentation, and packaging the results in a PDS-compliant data set. The data set is then validated and reviewed by a group of external scientists as part of the PDS final archive process. We present a status report on some of the data sets that we are processing.

ABMapper: a suffix array-based tool for multi-location searching and splice-junction mapping.

PubMed

Lou, Shao-Ke; Ni, Bing; Lo, Leung-Yau; Tsui, Stephen Kwok-Wing; Chan, Ting-Fung; Leung, Kwong-Sak

2011-02-01

Sequencing reads generated by RNA-sequencing (RNA-seq) must first be mapped back to the genome through alignment before they can be further analyzed. Current fast and memory-saving short-read mappers could give us a quick view of the transcriptome. However, they are neither designed for reads that span across splice junctions nor for repetitive reads, which can be mapped to multiple locations in the genome (multi-reads). Here, we describe a new software package: ABMapper, which is specifically designed for exploring all putative locations of reads that are mapped to splice junctions or repetitive in nature. The software is freely available at: http://abmapper.sourceforge.net/. The software is written in C++ and PERL. It runs on all major platforms and operating systems including Windows, Mac OS X and LINUX.

ShortRead: a bioconductor package for input, quality assessment and exploration of high-throughput sequence data

PubMed Central

Morgan, Martin; Anders, Simon; Lawrence, Michael; Aboyoun, Patrick; Pagès, Hervé; Gentleman, Robert

2009-01-01

Summary: ShortRead is a package for input, quality assessment, manipulation and output of high-throughput sequencing data. ShortRead is provided in the R and Bioconductor environments, allowing ready access to additional facilities for advanced statistical analysis, data transformation, visualization and integration with diverse genomic resources. Availability and Implementation: This package is implemented in R and available at the Bioconductor web site; the package contains a ‘vignette’ outlining typical work flows. Contact: mtmorgan@fhcrc.org PMID:19654119

An Artificial Functional Family Filter in Homolog Searching in Next-generation Sequencing Metagenomics

PubMed Central

Du, Ruofei; Mercante, Donald; Fang, Zhide

2013-01-01

In functional metagenomics, BLAST homology search is a common method to classify metagenomic reads into protein/domain sequence families such as Clusters of Orthologous Groups of proteins (COGs) in order to quantify the abundance of each COG in the community. The resulting functional profile of the community is then used in downstream analysis to correlate the change in abundance to environmental perturbation, clinical variation, and so on. However, the short read length coupled with next-generation sequencing technologies poses a barrier in this approach, essentially because similarity significance cannot be discerned by searching with short reads. Consequently, artificial functional families are produced, in which those with a large number of reads assigned decreases the accuracy of functional profile dramatically. There is no method available to address this problem. We intended to fill this gap in this paper. We revealed that BLAST similarity scores of homologues for short reads from COG protein members coding sequences are distributed differently from the scores of those derived elsewhere. We showed that, by choosing an appropriate score cut-off, we are able to filter out most artificial families and simultaneously to preserve sufficient information in order to build the functional profile. We also showed that, by incorporated application of BLAST and RPS-BLAST, some artificial families with large read counts can be further identified after the score cutoff filtration. Evaluated on three experimental metagenomic datasets with different coverages, we found that the proposed method is robust against read coverage and consistently outperforms the other E-value cutoff methods currently used in literatures. PMID:23516532

“An Adamless Eden” in Ingonish: what Cape Breton's archives reveal.

PubMed

Revie, Linda L

2010-01-01

This essay reads the archived life of a Sydney-based woman - Ella Liscombe (1902–69) - as it was recorded in her diaries, notebooks, and especially her photograph album of a 1927 camping excursion to Ingonish, Cape Breton Island. This album features pictures of women in "cross-dress," and the writings that gloss these camping records express Ella Liscombe’s erotic same-sex feelings about her female companions. As this essay explores Liscombe’s sartorial and emotional aesthetics, it also makes distinctions between "mannish" behaviour and "boyish" performance/costume, ultimately suggesting that Ella and her friends indulged in "twilight moments" to escape the strictures of domestic femininity.

Aided generation of search interfaces to astronomical archives

NASA Astrophysics Data System (ADS)

Zorba, Sonia; Bignamini, Andrea; Cepparo, Francesco; Knapic, Cristina; Molinaro, Marco; Smareglia, Riccardo

2016-07-01

Astrophysical data provider organizations that host web based interfaces to provide access to data resources have to cope with possible changes in data management that imply partial rewrites of web applications. To avoid doing this manually it was decided to develop a dynamically configurable Java EE web application that can set itself up reading needed information from configuration files. Specification of what information the astronomical archive database has to expose is managed using the TAP SCHEMA schema from the IVOA TAP recommendation, that can be edited using a graphical interface. When configuration steps are done the tool will build a war file to allow easy deployment of the application.

STORMSeq: An Open-Source, User-Friendly Pipeline for Processing Personal Genomics Data in the Cloud

PubMed Central

Karczewski, Konrad J.; Fernald, Guy Haskin; Martin, Alicia R.; Snyder, Michael; Tatonetti, Nicholas P.; Dudley, Joel T.

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5–10 hours to process a full exome sequence and $30 and 3–8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2. PMID:24454756

An investigation of causes of false positive single nucleotide polymorphisms using simulated reads from a small eukaryote genome.

PubMed

Ribeiro, Antonio; Golicz, Agnieszka; Hackett, Christine Anne; Milne, Iain; Stephen, Gordon; Marshall, David; Flavell, Andrew J; Bayer, Micha

2015-11-11

Single Nucleotide Polymorphisms (SNPs) are widely used molecular markers, and their use has increased massively since the inception of Next Generation Sequencing (NGS) technologies, which allow detection of large numbers of SNPs at low cost. However, both NGS data and their analysis are error-prone, which can lead to the generation of false positive (FP) SNPs. We explored the relationship between FP SNPs and seven factors involved in mapping-based variant calling - quality of the reference sequence, read length, choice of mapper and variant caller, mapping stringency and filtering of SNPs by read mapping quality and read depth. This resulted in 576 possible factor level combinations. We used error- and variant-free simulated reads to ensure that every SNP found was indeed a false positive. The variation in the number of FP SNPs generated ranged from 0 to 36,621 for the 120 million base pairs (Mbp) genome. All of the experimental factors tested had statistically significant effects on the number of FP SNPs generated and there was a considerable amount of interaction between the different factors. Using a fragmented reference sequence led to a dramatic increase in the number of FP SNPs generated, as did relaxed read mapping and a lack of SNP filtering. The choice of reference assembler, mapper and variant caller also significantly affected the outcome. The effect of read length was more complex and suggests a possible interaction between mapping specificity and the potential for contributing more false positives as read length increases. The choice of tools and parameters involved in variant calling can have a dramatic effect on the number of FP SNPs produced, with particularly poor combinations of software and/or parameter settings yielding tens of thousands in this experiment. Between-factor interactions make simple recommendations difficult for a SNP discovery pipeline but the quality of the reference sequence is clearly of paramount importance. Our findings are also a stark reminder that it can be unwise to use the relaxed mismatch settings provided as defaults by some read mappers when reads are being mapped to a relatively unfinished reference sequence from e.g. a non-model organism in its early stages of genomic exploration.

Comparison of next generation sequencing technologies for transcriptome characterization

PubMed Central

2009-01-01

Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms. PMID:19646272

Using the Tools and Resources of the RCSB Protein Data Bank.

PubMed

Costanzo, Luigi Di; Ghosh, Sutapa; Zardecki, Christine; Burley, Stephen K

2016-09-07

The Protein Data Bank (PDB) archive is the worldwide repository of experimentally determined three-dimensional structures of large biological molecules found in all three kingdoms of life. Atomic-level structures of these proteins, nucleic acids, and complex assemblies thereof are central to research and education in molecular, cellular, and organismal biology, biochemistry, biophysics, materials science, bioengineering, ecology, and medicine. Several types of information are associated with each PDB archival entry, including atomic coordinates, primary experimental data, polymer sequence(s), and summary metadata. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) serves as the U.S. data center for the PDB, distributing archival data and supporting both simple and complex queries that return results. These data can be freely downloaded, analyzed, and visualized using RCSB PDB tools and resources to gain a deeper understanding of fundamental biological processes, molecular evolution, human health and disease, and drug discovery. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Sakakibara, Yasumbumi

2018-02-13

Keio University's Yasumbumi Sakakibara on "MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

A Teaching-Learning Sequence about Weather Map Reading

ERIC Educational Resources Information Center

Mandrikas, Achilleas; Stavrou, Dimitrios; Skordoulis, Constantine

2017-01-01

In this paper a teaching-learning sequence (TLS) introducing pre-service elementary teachers (PET) to weather map reading, with emphasis on wind assignment, is presented. The TLS includes activities about recognition of wind symbols, assignment of wind direction and wind speed on a weather map and identification of wind characteristics in a…

MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakakibara, Yasumbumi

2011-10-13

Keio University's Yasumbumi Sakakibara on "MetaVelvet: An Extension of Velvet Assembler to de novo Metagenome Assembly from Short Sequence Reads" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Finished Prokaryotic Genome Assemblies from a Low-cost Combination of Short and Long Reads (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, Shuangye

2012-06-01

Shuangye Yin on "Finished prokaryotic genome assemblies from a low-cost combination of short and long reads"; at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

AFRESh: an adaptive framework for compression of reads and assembled sequences with random access functionality.

PubMed

Paridaens, Tom; Van Wallendael, Glenn; De Neve, Wesley; Lambert, Peter

2017-05-15

The past decade has seen the introduction of new technologies that lowered the cost of genomic sequencing increasingly. We can even observe that the cost of sequencing is dropping significantly faster than the cost of storage and transmission. The latter motivates a need for continuous improvements in the area of genomic data compression, not only at the level of effectiveness (compression rate), but also at the level of functionality (e.g. random access), configurability (effectiveness versus complexity, coding tool set …) and versatility (support for both sequenced reads and assembled sequences). In that regard, we can point out that current approaches mostly do not support random access, requiring full files to be transmitted, and that current approaches are restricted to either read or sequence compression. We propose AFRESh, an adaptive framework for no-reference compression of genomic data with random access functionality, targeting the effective representation of the raw genomic symbol streams of both reads and assembled sequences. AFRESh makes use of a configurable set of prediction and encoding tools, extended by a Context-Adaptive Binary Arithmetic Coding scheme (CABAC), to compress raw genetic codes. To the best of our knowledge, our paper is the first to describe an effective implementation CABAC outside of its' original application. By applying CABAC, the compression effectiveness improves by up to 19% for assembled sequences and up to 62% for reads. By applying AFRESh to the genomic symbols of the MPEG genomic compression test set for reads, a compression gain is achieved of up to 51% compared to SCALCE, 42% compared to LFQC and 44% compared to ORCOM. When comparing to generic compression approaches, a compression gain is achieved of up to 41% compared to GNU Gzip and 22% compared to 7-Zip at the Ultra setting. Additionaly, when compressing assembled sequences of the Human Genome, a compression gain is achieved up to 34% compared to GNU Gzip and 16% compared to 7-Zip at the Ultra setting. A Windows executable version can be downloaded at https://github.com/tparidae/AFresh . tom.paridaens@ugent.be. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

ADEPT, a dynamic next generation sequencing data error-detection program with trimming

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng, Shihai; Lo, Chien-Chi; Li, Po-E

Illumina is the most widely used next generation sequencing technology and produces millions of short reads that contain errors. These sequencing errors constitute a major problem in applications such as de novo genome assembly, metagenomics analysis and single nucleotide polymorphism discovery. In this study, we present ADEPT, a dynamic error detection method, based on the quality scores of each nucleotide and its neighboring nucleotides, together with their positions within the read and compares this to the position-specific quality score distribution of all bases within the sequencing run. This method greatly improves upon other available methods in terms of the truemore » positive rate of error discovery without affecting the false positive rate, particularly within the middle of reads. We conclude that ADEPT is the only tool to date that dynamically assesses errors within reads by comparing position-specific and neighboring base quality scores with the distribution of quality scores for the dataset being analyzed. The result is a method that is less prone to position-dependent under-prediction, which is one of the most prominent issues in error prediction. The outcome is that ADEPT improves upon prior efforts in identifying true errors, primarily within the middle of reads, while reducing the false positive rate.« less

ADEPT, a dynamic next generation sequencing data error-detection program with trimming

DOE PAGES

Feng, Shihai; Lo, Chien-Chi; Li, Po-E; ...

2016-02-29

Illumina is the most widely used next generation sequencing technology and produces millions of short reads that contain errors. These sequencing errors constitute a major problem in applications such as de novo genome assembly, metagenomics analysis and single nucleotide polymorphism discovery. In this study, we present ADEPT, a dynamic error detection method, based on the quality scores of each nucleotide and its neighboring nucleotides, together with their positions within the read and compares this to the position-specific quality score distribution of all bases within the sequencing run. This method greatly improves upon other available methods in terms of the truemore » positive rate of error discovery without affecting the false positive rate, particularly within the middle of reads. We conclude that ADEPT is the only tool to date that dynamically assesses errors within reads by comparing position-specific and neighboring base quality scores with the distribution of quality scores for the dataset being analyzed. The result is a method that is less prone to position-dependent under-prediction, which is one of the most prominent issues in error prediction. The outcome is that ADEPT improves upon prior efforts in identifying true errors, primarily within the middle of reads, while reducing the false positive rate.« less

Functional sequencing read annotation for high precision microbiome analysis

PubMed Central

Zhu, Chengsheng; Miller, Maximilian; Marpaka, Srinayani; Vaysberg, Pavel; Rühlemann, Malte C; Wu, Guojun; Heinsen, Femke-Anouska; Tempel, Marie; Zhao, Liping; Lieb, Wolfgang; Franke, Andre; Bromberg, Yana

2018-01-01

Abstract The vast majority of microorganisms on Earth reside in often-inseparable environment-specific communities—microbiomes. Meta-genomic/-transcriptomic sequencing could reveal the otherwise inaccessible functionality of microbiomes. However, existing analytical approaches focus on attributing sequencing reads to known genes/genomes, often failing to make maximal use of available data. We created faser (functional annotation of sequencing reads), an algorithm that is optimized to map reads to molecular functions encoded by the read-correspondent genes. The mi-faser microbiome analysis pipeline, combining faser with our manually curated reference database of protein functions, accurately annotates microbiome molecular functionality. mi-faser’s minutes-per-microbiome processing speed is significantly faster than that of other methods, allowing for large scale comparisons. Microbiome function vectors can be compared between different conditions to highlight environment-specific and/or time-dependent changes in functionality. Here, we identified previously unseen oil degradation-specific functions in BP oil-spill data, as well as functional signatures of individual-specific gut microbiome responses to a dietary intervention in children with Prader–Willi syndrome. Our method also revealed variability in Crohn's Disease patient microbiomes and clearly distinguished them from those of related healthy individuals. Our analysis highlighted the microbiome role in CD pathogenicity, demonstrating enrichment of patient microbiomes in functions that promote inflammation and that help bacteria survive it. PMID:29194524

High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis

PubMed Central

Arron, Sarah T.; Dimon, Michelle T.; Li, Zhenghui; Johnson, Michael E.; Wood, Tammara; Feeney, Luzviminda; Angeles, Jorge Gil; Lafyatis, Robert; Whitfield, Michael L.

2014-01-01

Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), though neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared to normal controls. RNA-seq was performed on four SSc and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the non-human sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1–D2 hypervariable region of the 28S rRNA of R. glutinis from each of the SSc samples. Taken together, these results suggest R. glutinis may be present in the skin of early SSc patients at higher levels than normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc. PMID:24608988

High Rhodotorula sequences in skin transcriptome of patients with diffuse systemic sclerosis.

PubMed

Arron, Sarah T; Dimon, Michelle T; Li, Zhenghui; Johnson, Michael E; Wood, Tammara A; Feeney, Luzviminda; Angeles, Jorge G; Lafyatis, Robert; Whitfield, Michael L

2014-08-01

Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), although neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared with that in normal controls. RNA-seq was performed on four SSc patients and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the nonhuman sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1-D2 hypervariable region of the 28S ribosomal RNA (rRNA) of R. glutinis from each of the SSc samples. Taken together, these results suggest that R. glutinis may be present in the skin of early SSc patients at higher levels than in normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc.

MALINA: a web service for visual analytics of human gut microbiota whole-genome metagenomic reads.

PubMed

Tyakht, Alexander V; Popenko, Anna S; Belenikin, Maxim S; Altukhov, Ilya A; Pavlenko, Alexander V; Kostryukova, Elena S; Selezneva, Oksana V; Larin, Andrei K; Karpova, Irina Y; Alexeev, Dmitry G

2012-12-07

MALINA is a web service for bioinformatic analysis of whole-genome metagenomic data obtained from human gut microbiota sequencing. As input data, it accepts metagenomic reads of various sequencing technologies, including long reads (such as Sanger and 454 sequencing) and next-generation (including SOLiD and Illumina). It is the first metagenomic web service that is capable of processing SOLiD color-space reads, to authors' knowledge. The web service allows phylogenetic and functional profiling of metagenomic samples using coverage depth resulting from the alignment of the reads to the catalogue of reference sequences which are built into the pipeline and contain prevalent microbial genomes and genes of human gut microbiota. The obtained metagenomic composition vectors are processed by the statistical analysis and visualization module containing methods for clustering, dimension reduction and group comparison. Additionally, the MALINA database includes vectors of bacterial and functional composition for human gut microbiota samples from a large number of existing studies allowing their comparative analysis together with user samples, namely datasets from Russian Metagenome project, MetaHIT and Human Microbiome Project (downloaded from http://hmpdacc.org). MALINA is made freely available on the web at http://malina.metagenome.ru. The website is implemented in JavaScript (using Ext JS), Microsoft .NET Framework, MS SQL, Python, with all major browsers supported.

Benefits of cloud computing for PACS and archiving.

PubMed

Koch, Patrick

2012-01-01

The goal of cloud-based services is to provide easy, scalable access to computing resources and IT services. The healthcare industry requires a private cloud that adheres to government mandates designed to ensure privacy and security of patient data while enabling access by authorized users. Cloud-based computing in the imaging market has evolved from a service that provided cost effective disaster recovery for archived data to fully featured PACS and vendor neutral archiving services that can address the needs of healthcare providers of all sizes. Healthcare providers worldwide are now using the cloud to distribute images to remote radiologists while supporting advanced reading tools, deliver radiology reports and imaging studies to referring physicians, and provide redundant data storage. Vendor managed cloud services eliminate large capital investments in equipment and maintenance, as well as staffing for the data center--creating a reduction in total cost of ownership for the healthcare provider.

A search for T Tauri stars and related objects: Archival photometry of candidate variables in V733 Cep field

NASA Astrophysics Data System (ADS)

Jurdana-Šepić, R.; Poljančić Beljan, I.

Searching for T Tauri stars or related early type variables we carried out a BVRI photometric measurements of five candidates with positions within the field of the pre-main sequence object V733 Cephei (Persson's star) located in the dark cloud L1216 near to Cepheus OB3 Association: VES 946, VES 950, NSV 14333, NSV 25966 and V385 Cep. Their magnitudes are determined on the plates from Asiago Observatory historical photographic archive exposed 1971 - 1978. We provide finding charts for program stars and comparison sequence stars, magnitude estimations, magnitude mean values and BVR_cI_c light curves of program stars.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

Performance of children with typical development when reading and interpreting graphic-symbol sequences.

PubMed

Boyer, Catherine; Trudeau, Natacha; Sutton, Ann

2012-06-01

In order to understand a sequence of graphic symbols as sentences, one must not only recognize the meaning of individual symbols but also integrate their meaning together. In this study children without disabilities were asked to perform two tasks that presented sequences of graphics as stimuli but that differed in the need to treat the symbols as a sentence (i.e., with evidence of relationships among the individual symbols): a "reading" task (transpose the symbol sequence into speech), and an act-out task (demonstrate the meaning of the symbol sequences using puppets). The participants, aged 3 (n=18), 4 (n=36), 5 (n=27), and 6 (n=23) years, all succeeded on the reading task, but the younger groups were much less successful than the older groups on the act-out task. The children were more likely to pass the act-out task if they used conjugated rather than infinitive verb forms in their spoken responses on the reading task. In the younger age groups, children who used conjugated verb forms had higher receptive vocabulary scores. The findings suggest that being able to reproduce a sequence of symbols does not guarantee that the symbols are treated as a sentence. The inclusion in the study of children who were able to respond using speech, permitted observation of two types of responses (conjugated versus infinitive verb forms) that revealed different levels of understanding of graphic symbol sequences.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data.

PubMed

Li, Ruichao; Xie, Miaomiao; Dong, Ning; Lin, Dachuan; Yang, Xuemei; Wong, Marcus Ho Yin; Chan, Edward Wai-Chi; Chen, Sheng

2018-03-01

Multidrug resistance (MDR)-encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming. Here, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy. This workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.

Detecting exact breakpoints of deletions with diversity in hepatitis B viral genomic DNA from next-generation sequencing data.

PubMed

Cheng, Ji-Hong; Liu, Wen-Chun; Chang, Ting-Tsung; Hsieh, Sun-Yuan; Tseng, Vincent S

2017-10-01

Many studies have suggested that deletions of Hepatitis B Viral (HBV) are associated with the development of progressive liver diseases, even ultimately resulting in hepatocellular carcinoma (HCC). Among the methods for detecting deletions from next-generation sequencing (NGS) data, few methods considered the characteristics of virus, such as high evolution rates and high divergence among the different HBV genomes. Sequencing high divergence HBV genome sequences using the NGS technology outputs millions of reads. Thus, detecting exact breakpoints of deletions from these big and complex data incurs very high computational cost. We proposed a novel analytical method named VirDelect (Virus Deletion Detect), which uses split read alignment base to detect exact breakpoint and diversity variable to consider high divergence in single-end reads data, such that the computational cost can be reduced without losing accuracy. We use four simulated reads datasets and two real pair-end reads datasets of HBV genome sequence to verify VirDelect accuracy by score functions. The experimental results show that VirDelect outperforms the state-of-the-art method Pindel in terms of accuracy score for all simulated datasets and VirDelect had only two base errors even in real datasets. VirDelect is also shown to deliver high accuracy in analyzing the single-end read data as well as pair-end data. VirDelect can serve as an effective and efficient bioinformatics tool for physiologists with high accuracy and efficient performance and applicable to further analysis with characteristics similar to HBV on genome length and high divergence. The software program of VirDelect can be downloaded at https://sourceforge.net/projects/virdelect/. Copyright © 2017. Published by Elsevier Inc.

Expressed sequence tags from heat-shocked seagrass Zostera noltii (Hornemann) from its southern distribution range.

PubMed

Massa, Sónia I; Pearson, Gareth A; Aires, Tânia; Kube, Michael; Olsen, Jeanine L; Reinhardt, Richard; Serrão, Ester A; Arnaud-Haond, Sophie

2011-09-01

Predicted global climate change threatens the distributional ranges of species worldwide. We identified genes expressed in the intertidal seagrass Zostera noltii during recovery from a simulated low tide heat-shock exposure. Five Expressed Sequence Tag (EST) libraries were compared, corresponding to four recovery times following sub-lethal temperature stress, and a non-stressed control. We sequenced and analyzed 7009 sequence reads from 30min, 2h, 4h and 24h after the beginning of the heat-shock (AHS), and 1585 from the control library, for a total of 8594 sequence reads. Among 51 Tentative UniGenes (TUGs) exhibiting significantly different expression between libraries, 19 (37.3%) were identified as 'molecular chaperones' and were over-expressed following heat-shock, while 12 (23.5%) were 'photosynthesis TUGs' generally under-expressed in heat-shocked plants. A time course analysis of expression showed a rapid increase in expression of the molecular chaperone class, most of which were heat-shock proteins; which increased from 2 sequence reads in the control library to almost 230 in the 30min AHS library, followed by a slow decrease during further recovery. In contrast, 'photosynthesis TUGs' were under-expressed 30min AHS compared with the control library, and declined progressively with recovery time in the stress libraries, with a total of 29 sequence reads 24h AHS, compared with 125 in the control. A total of 4734 TUGs were screened for EST-Single Sequence Repeats (EST-SSRs) and 86 microsatellites were identified. Copyright © 2011 Elsevier B.V. All rights reserved.

Curated eutherian third party data gene data sets.

PubMed

Premzl, Marko

2016-03-01

The free available eutherian genomic sequence data sets advanced scientific field of genomics. Of note, future revisions of gene data sets were expected, due to incompleteness of public eutherian genomic sequence assemblies and potential genomic sequence errors. The eutherian comparative genomic analysis protocol was proposed as guidance in protection against potential genomic sequence errors in public eutherian genomic sequences. The protocol was applicable in updates of 7 major eutherian gene data sets, including 812 complete coding sequences deposited in European Nucleotide Archive as curated third party data gene data sets.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

PubMed

Watson, Christopher M; Camm, Nick; Crinnion, Laura A; Clokie, Samuel; Robinson, Rachel L; Adlard, Julian; Charlton, Ruth; Markham, Alexander F; Carr, Ian M; Bonthron, David T

2017-12-01

Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.

NGSPanPipe: A Pipeline for Pan-genome Identification in Microbial Strains from Experimental Reads.

PubMed

Kulsum, Umay; Kapil, Arti; Singh, Harpreet; Kaur, Punit

2018-01-01

Recent advancements in sequencing technologies have decreased both time span and cost for sequencing the whole bacterial genome. High-throughput Next-Generation Sequencing (NGS) technology has led to the generation of enormous data concerning microbial populations publically available across various repositories. As a consequence, it has become possible to study and compare the genomes of different bacterial strains within a species or genus in terms of evolution, ecology and diversity. Studying the pan-genome provides insights into deciphering microevolution, global composition and diversity in virulence and pathogenesis of a species. It can also assist in identifying drug targets and proposing vaccine candidates. The effective analysis of these large genome datasets necessitates the development of robust tools. Current methods to develop pan-genome do not support direct input of raw reads from the sequencer machine but require preprocessing of reads as an assembled protein/gene sequence file or the binary matrix of orthologous genes/proteins. We have designed an easy-to-use integrated pipeline, NGSPanPipe, which can directly identify the pan-genome from short reads. The output from the pipeline is compatible with other pan-genome analysis tools. We evaluated our pipeline with other methods for developing pan-genome, i.e. reference-based assembly and de novo assembly using simulated reads of Mycobacterium tuberculosis. The single script pipeline (pipeline.pl) is applicable for all bacterial strains. It integrates multiple in-house Perl scripts and is freely accessible from https://github.com/Biomedinformatics/NGSPanPipe .

An Outbreak of Respiratory Tularemia Caused by Diverse Clones of Francisella tularensis

PubMed Central

Johansson, Anders; Lärkeryd, Adrian; Widerström, Micael; Mörtberg, Sara; Myrtännäs, Kerstin; Öhrman, Caroline; Birdsell, Dawn; Keim, Paul; Wagner, David M.; Forsman, Mats; Larsson, Pär

2014-01-01

Background. The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. Methods. A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. Results. There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1–3 of 1 585 229 examined nucleotides. Conclusions. The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link. PMID:25097081

The Scientisation of Schooling in Ontario, 1910-1934

ERIC Educational Resources Information Center

Milewski, Patrice

2010-01-01

This paper analyses the science of education that was formed in Ontario between the years 1910 and 1934. It is substantiated through the use of archival material such as curriculum documents, statutes, annual reports, the published proceedings of the Ontario Educational Association (OEA) and a close reading of the "Science of Education"…

Internet-Based Cervical Cancer Screening Program

DTIC Science & Technology

2008-05-01

information technology have facilitated the Internet transmission and archival storage of digital images and other clinical information . The combination of...Phase included: 1) development of hardware, software, and interfaces between computerized scanning device and Internet - linked servers and reading...AD_________________ Award Number: W81XWH-04-C-0083 TITLE: Internet -Based Cervical Cancer Screening

"The BFG" and the Spaghetti Book Club: A Case Study of Children as Critics

ERIC Educational Resources Information Center

Hoffman, A. Robin

2010-01-01

Situated at the intersections of ethnography, childhood studies, literary studies, and education research, this reception study seeks to access real children's responses to a particular text, and to offer empirical description of actual reading experiences. Survey data is generated by taking advantage of an online resource: an archive of…

Underachievement in Primary Grade Students: A Review of Kindergarten Enrollment and DIBELS Scores

ERIC Educational Resources Information Center

Rice, Shawnik Marie

2013-01-01

Student underachievement in kindergarten through Grade 3 continues to be a challenge in the Philadelphia School District. The purpose of this quantitative descriptive correlation study was to examine, using record archives from one Philadelphia school, whether there is a relationship between (a) reading achievement scores for the Dynamic…

36 CFR 1235.46 - What electronic media may be used for transferring records to the National Archives of the United...

Code of Federal Regulations, 2012 CFR

2012-07-01

... that meet ANSI X3.39 or ANSI X3.54 (both incorporated by reference, see § 1235.4), respectively. (2) 18...) Compact-Disk, Read Only Memory (CD-ROM) and Digital Video Disks (DVDs). Agencies may use CD-ROMs and DVDs...

36 CFR 1235.46 - What electronic media may be used for transferring records to the National Archives of the United...

Code of Federal Regulations, 2010 CFR

2010-07-01

... that meet ANSI X3.39 or ANSI X3.54 (both incorporated by reference, see § 1235.4), respectively. (2) 18...) Compact-Disk, Read Only Memory (CD-ROM) and Digital Video Disks (DVDs). Agencies may use CD-ROMs and DVDs...

36 CFR 1235.46 - What electronic media may be used for transferring records to the National Archives of the United...

Code of Federal Regulations, 2014 CFR

2014-07-01

... that meet ANSI X3.39 or ANSI X3.54 (both incorporated by reference, see § 1235.4), respectively. (2) 18...) Compact-Disk, Read Only Memory (CD-ROM) and Digital Video Disks (DVDs). Agencies may use CD-ROMs and DVDs...

36 CFR 1235.46 - What electronic media may be used for transferring records to the National Archives of the United...

Code of Federal Regulations, 2011 CFR

2011-07-01

... that meet ANSI X3.39 or ANSI X3.54 (both incorporated by reference, see § 1235.4), respectively. (2) 18...) Compact-Disk, Read Only Memory (CD-ROM) and Digital Video Disks (DVDs). Agencies may use CD-ROMs and DVDs...

36 CFR § 1235.46 - What electronic media may be used for transferring records to the National Archives of the United...

Code of Federal Regulations, 2013 CFR

2013-07-01

...), respectively. (2) 18-track 3480-class cartridges must be recorded at 37,871 bpi that meet ANSI X3.180..., § 1235.4). (c) Compact-Disk, Read Only Memory (CD-ROM) and Digital Video Disks (DVDs). Agencies may use...

Textual Encounters in the DALN/"Composition Forum" on the DALN

ERIC Educational Resources Information Center

Soliday, Mary

2017-01-01

In this article, Mary Soliday discusses her observation that within the Digital Archive of Literacy Narratives (DALN), the narrators frequently attribute their desire to read and write in the present with specific textual encounters they had had in the past. In these encounters, the text (often a literary text) helped narrators to mediate…

Computers and Computation. Readings from Scientific American.

ERIC Educational Resources Information Center

Fenichel, Robert R.; Weizenbaum, Joseph

A collection of articles from "Scientific American" magazine has been put together at this time because the current period in computer science is one of consolidation rather than innovation. A few years ago, computer science was moving so swiftly that even the professional journals were more archival than informative; but today it is…

The Challenges Facing Science Data Archiving on Current Mass Storage Systems

NASA Technical Reports Server (NTRS)

Peavey, Bernard; Behnke, Jeanne (Editor)

1996-01-01

This paper discusses the desired characteristics of a tape-based petabyte science data archive and retrieval system required to store and distribute several terabytes (TB) of data per day over an extended period of time, probably more than 115 years, in support of programs such as the Earth Observing System Data and Information System (EOSDIS). These characteristics take into consideration not only cost effective and affordable storage capacity, but also rapid access to selected files, and reading rates that are needed to satisfy thousands of retrieval transactions per day. It seems that where rapid random access to files is not crucial, the tape medium, magnetic or optical, continues to offer cost effective data storage and retrieval solutions, and is likely to do so for many years to come. However, in environments like EOS these tape based archive solutions provide less than full user satisfaction. Therefore, the objective of this paper is to describe the performance and operational enhancements that need to be made to the current tape based archival systems in order to achieve greater acceptance by the EOS and similar user communities.

Plant Omics Data Center: An Integrated Web Repository for Interspecies Gene Expression Networks with NLP-Based Curation

PubMed Central

Ohyanagi, Hajime; Takano, Tomoyuki; Terashima, Shin; Kobayashi, Masaaki; Kanno, Maasa; Morimoto, Kyoko; Kanegae, Hiromi; Sasaki, Yohei; Saito, Misa; Asano, Satomi; Ozaki, Soichi; Kudo, Toru; Yokoyama, Koji; Aya, Koichiro; Suwabe, Keita; Suzuki, Go; Aoki, Koh; Kubo, Yasutaka; Watanabe, Masao; Matsuoka, Makoto; Yano, Kentaro

2015-01-01

Comprehensive integration of large-scale omics resources such as genomes, transcriptomes and metabolomes will provide deeper insights into broader aspects of molecular biology. For better understanding of plant biology, we aim to construct a next-generation sequencing (NGS)-derived gene expression network (GEN) repository for a broad range of plant species. So far we have incorporated information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max) from the public short read archive, digitally profiled the entire set of gene expression profiles, and drawn GENs by using correspondence analysis (CA) to take advantage of gene expression similarities. In order to understand the evolutionary significance of the GENs from multiple species, they were linked according to the orthology of each node (gene) among species. In addition to other gene expression information, functional annotation of the genes will facilitate biological comprehension. Currently we are improving the given gene annotations with natural language processing (NLP) techniques and manual curation. Here we introduce the current status of our analyses and the web database, PODC (Plant Omics Data Center; http://bioinf.mind.meiji.ac.jp/podc/), now open to the public, providing GENs, functional annotations and additional comprehensive omics resources. PMID:25505034

Graph mining for next generation sequencing: leveraging the assembly graph for biological insights.

PubMed

Warnke-Sommer, Julia; Ali, Hesham

2016-05-06

The assembly of Next Generation Sequencing (NGS) reads remains a challenging task. This is especially true for the assembly of metagenomics data that originate from environmental samples potentially containing hundreds to thousands of unique species. The principle objective of current assembly tools is to assemble NGS reads into contiguous stretches of sequence called contigs while maximizing for both accuracy and contig length. The end goal of this process is to produce longer contigs with the major focus being on assembly only. Sequence read assembly is an aggregative process, during which read overlap relationship information is lost as reads are merged into longer sequences or contigs. The assembly graph is information rich and capable of capturing the genomic architecture of an input read data set. We have developed a novel hybrid graph in which nodes represent sequence regions at different levels of granularity. This model, utilized in the assembly and analysis pipeline Focus, presents a concise yet feature rich view of a given input data set, allowing for the extraction of biologically relevant graph structures for graph mining purposes. Focus was used to create hybrid graphs to model metagenomics data sets obtained from the gut microbiomes of five individuals with Crohn's disease and eight healthy individuals. Repetitive and mobile genetic elements are found to be associated with hybrid graph structure. Using graph mining techniques, a comparative study of the Crohn's disease and healthy data sets was conducted with focus on antibiotics resistance genes associated with transposase genes. Results demonstrated significant differences in the phylogenetic distribution of categories of antibiotics resistance genes in the healthy and diseased patients. Focus was also evaluated as a pure assembly tool and produced excellent results when compared against the Meta-velvet, Omega, and UD-IDBA assemblers. Mining the hybrid graph can reveal biological phenomena captured by its structure. We demonstrate the advantages of considering assembly graphs as data-mining support in addition to their role as frameworks for assembly.

Comparison of the Equine Reference Sequence with Its Sanger Source Data and New Illumina Reads

PubMed Central

Rebolledo-Mendez, Jovan; Hestand, Matthew S.; Coleman, Stephen J.; Zeng, Zheng; Orlando, Ludovic; MacLeod, James N.; Kalbfleisch, Ted

2015-01-01

The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight’s half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects’ and Twilight’s genome or due to errors in the reference. EquCab2 is regarded as “The Twilight Assembly.” The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies between new, high throughput genomic sequence data generated for Twilight and the EquCab2 reference assembly. Most of these represent errors in the assembly, while approximately 10,000 are demonstrated to be contributions from another horse. Other results are presented that include the binary alignment map file of the mapped Sanger reads, a list of variants identified as discrepancies between the source data and resulting reference, and a BED annotation file that lists the regions of the genome whose consensus was likely derived from low coverage alignments. PMID:26107638

Model-based quality assessment and base-calling for second-generation sequencing data.

PubMed

Bravo, Héctor Corrada; Irizarry, Rafael A

2010-09-01

Second-generation sequencing (sec-gen) technology can sequence millions of short fragments of DNA in parallel, making it capable of assembling complex genomes for a small fraction of the price and time of previous technologies. In fact, a recently formed international consortium, the 1000 Genomes Project, plans to fully sequence the genomes of approximately 1200 people. The prospect of comparative analysis at the sequence level of a large number of samples across multiple populations may be achieved within the next five years. These data present unprecedented challenges in statistical analysis. For instance, analysis operates on millions of short nucleotide sequences, or reads-strings of A,C,G, or T's, between 30 and 100 characters long-which are the result of complex processing of noisy continuous fluorescence intensity measurements known as base-calling. The complexity of the base-calling discretization process results in reads of widely varying quality within and across sequence samples. This variation in processing quality results in infrequent but systematic errors that we have found to mislead downstream analysis of the discretized sequence read data. For instance, a central goal of the 1000 Genomes Project is to quantify across-sample variation at the single nucleotide level. At this resolution, small error rates in sequencing prove significant, especially for rare variants. Sec-gen sequencing is a relatively new technology for which potential biases and sources of obscuring variation are not yet fully understood. Therefore, modeling and quantifying the uncertainty inherent in the generation of sequence reads is of utmost importance. In this article, we present a simple model to capture uncertainty arising in the base-calling procedure of the Illumina/Solexa GA platform. Model parameters have a straightforward interpretation in terms of the chemistry of base-calling allowing for informative and easily interpretable metrics that capture the variability in sequencing quality. Our model provides these informative estimates readily usable in quality assessment tools while significantly improving base-calling performance. © 2009, The International Biometric Society.

ArrayExpress update--trends in database growth and links to data analysis tools.

PubMed

Rustici, Gabriella; Kolesnikov, Nikolay; Brandizi, Marco; Burdett, Tony; Dylag, Miroslaw; Emam, Ibrahim; Farne, Anna; Hastings, Emma; Ison, Jon; Keays, Maria; Kurbatova, Natalja; Malone, James; Mani, Roby; Mupo, Annalisa; Pedro Pereira, Rui; Pilicheva, Ekaterina; Rung, Johan; Sharma, Anjan; Tang, Y Amy; Ternent, Tobias; Tikhonov, Andrew; Welter, Danielle; Williams, Eleanor; Brazma, Alvis; Parkinson, Helen; Sarkans, Ugis

2013-01-01

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is one of three international functional genomics public data repositories, alongside the Gene Expression Omnibus at NCBI and the DDBJ Omics Archive, supporting peer-reviewed publications. It accepts data generated by sequencing or array-based technologies and currently contains data from almost a million assays, from over 30 000 experiments. The proportion of sequencing-based submissions has grown significantly over the last 2 years and has reached, in 2012, 15% of all new data. All data are available from ArrayExpress in MAGE-TAB format, which allows robust linking to data analysis and visualization tools, including Bioconductor and GenomeSpace. Additionally, R objects, for microarray data, and binary alignment format files, for sequencing data, have been generated for a significant proportion of ArrayExpress data.

MetaCAA: A clustering-aided methodology for efficient assembly of metagenomic datasets.

PubMed

Reddy, Rachamalla Maheedhar; Mohammed, Monzoorul Haque; Mande, Sharmila S

2014-01-01

A key challenge in analyzing metagenomics data pertains to assembly of sequenced DNA fragments (i.e. reads) originating from various microbes in a given environmental sample. Several existing methodologies can assemble reads originating from a single genome. However, these methodologies cannot be applied for efficient assembly of metagenomic sequence datasets. In this study, we present MetaCAA - a clustering-aided methodology which helps in improving the quality of metagenomic sequence assembly. MetaCAA initially groups sequences constituting a given metagenome into smaller clusters. Subsequently, sequences in each cluster are independently assembled using CAP3, an existing single genome assembly program. Contigs formed in each of the clusters along with the unassembled reads are then subjected to another round of assembly for generating the final set of contigs. Validation using simulated and real-world metagenomic datasets indicates that MetaCAA aids in improving the overall quality of assembly. A software implementation of MetaCAA is available at https://metagenomics.atc.tcs.com/MetaCAA. Copyright © 2014 Elsevier Inc. All rights reserved.

Illuminator, a desktop program for mutation detection using short-read clonal sequencing.

PubMed

Carr, Ian M; Morgan, Joanne E; Diggle, Christine P; Sheridan, Eamonn; Markham, Alexander F; Logan, Clare V; Inglehearn, Chris F; Taylor, Graham R; Bonthron, David T

2011-10-01

Current methods for sequencing clonal populations of DNA molecules yield several gigabases of data per day, typically comprising reads of < 100 nt. Such datasets permit widespread genome resequencing and transcriptome analysis or other quantitative tasks. However, this huge capacity can also be harnessed for the resequencing of smaller (gene-sized) target regions, through the simultaneous parallel analysis of multiple subjects, using sample "tagging" or "indexing". These methods promise to have a huge impact on diagnostic mutation analysis and candidate gene testing. Here we describe a software package developed for such studies, offering the ability to resolve pooled samples carrying barcode tags and to align reads to a reference sequence using a mutation-tolerant process. The program, Illuminator, can identify rare sequence variants, including insertions and deletions, and permits interactive data analysis on standard desktop computers. It facilitates the effective analysis of targeted clonal sequencer data without dedicated computational infrastructure or specialized training. Copyright © 2011 Elsevier Inc. All rights reserved.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes

PubMed Central

2011-01-01

Background We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. Results The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. Conclusions BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies. PMID:21496274

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity

PubMed Central

Hurst, Gregory D.D.

2017-01-01

High throughput (or ‘next generation’) sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and ‘contaminating’ material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these ‘contaminations’ provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee (Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo. We conclude that ‘contamination’ in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses. PMID:28717593

Identification and correction of systematic error in high-throughput sequence data

PubMed Central

2011-01-01

Background A feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. The implications of such errors are application specific, ranging from minor informatics nuisances to major problems affecting biological inferences. Recently developed "next-gen" sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. We describe a new type of systematic error that manifests as statistically unlikely accumulations of errors at specific genome (or transcriptome) locations. Results We characterize and describe systematic errors using overlapping paired reads from high-coverage data. We show that such errors occur in approximately 1 in 1000 base pairs, and that they are highly replicable across experiments. We identify motifs that are frequent at systematic error sites, and describe a classifier that distinguishes heterozygous sites from systematic error. Our classifier is designed to accommodate data from experiments in which the allele frequencies at heterozygous sites are not necessarily 0.5 (such as in the case of RNA-Seq), and can be used with single-end datasets. Conclusions Systematic errors can easily be mistaken for heterozygous sites in individuals, or for SNPs in population analyses. Systematic errors are particularly problematic in low coverage experiments, or in estimates of allele-specific expression from RNA-Seq data. Our characterization of systematic error has allowed us to develop a program, called SysCall, for identifying and correcting such errors. We conclude that correction of systematic errors is important to consider in the design and interpretation of high-throughput sequencing experiments. PMID:22099972

Short reads from honey bee (Apis sp.) sequencing projects reflect microbial associate diversity.

PubMed

Gerth, Michael; Hurst, Gregory D D

2017-01-01

High throughput (or 'next generation') sequencing has transformed most areas of biological research and is now a standard method that underpins empirical study of organismal biology, and (through comparison of genomes), reveals patterns of evolution. For projects focused on animals, these sequencing methods do not discriminate between the primary target of sequencing (the animal genome) and 'contaminating' material, such as associated microbes. A common first step is to filter out these contaminants to allow better assembly of the animal genome or transcriptome. Here, we aimed to assess if these 'contaminations' provide information with regard to biologically important microorganisms associated with the individual. To achieve this, we examined whether the short read data from Apis retrieved elements of its well established microbiome. To this end, we screened almost 1,000 short read libraries of honey bee ( Apis sp.) DNA sequencing project for the presence of microbial sequences, and find sequences from known honey bee microbial associates in at least 11% of them. Further to this, we screened ∼500 Apis RNA sequencing libraries for evidence of viral infections, which were found to be present in about half of them. We then used the data to reconstruct draft genomes of three Apis associated bacteria, as well as several viral strains de novo . We conclude that 'contamination' in short read sequencing libraries can provide useful genomic information on microbial taxa known to be associated with the target organisms, and may even lead to the discovery of novel associations. Finally, we demonstrate that RNAseq samples from experiments commonly carry uneven viral loads across libraries. We note variation in viral presence and load may be a confounding feature of differential gene expression analyses, and as such it should be incorporated as a random factor in analyses.

zUMIs - A fast and flexible pipeline to process RNA sequencing data with UMIs.

PubMed

Parekh, Swati; Ziegenhain, Christoph; Vieth, Beate; Enard, Wolfgang; Hellmann, Ines

2018-06-01

Single-cell RNA-sequencing (scRNA-seq) experiments typically analyze hundreds or thousands of cells after amplification of the cDNA. The high throughput is made possible by the early introduction of sample-specific bar codes (BCs), and the amplification bias is alleviated by unique molecular identifiers (UMIs). Thus, the ideal analysis pipeline for scRNA-seq data needs to efficiently tabulate reads according to both BC and UMI. zUMIs is a pipeline that can handle both known and random BCs and also efficiently collapse UMIs, either just for exon mapping reads or for both exon and intron mapping reads. If BC annotation is missing, zUMIs can accurately detect intact cells from the distribution of sequencing reads. Another unique feature of zUMIs is the adaptive downsampling function that facilitates dealing with hugely varying library sizes but also allows the user to evaluate whether the library has been sequenced to saturation. To illustrate the utility of zUMIs, we analyzed a single-nucleus RNA-seq dataset and show that more than 35% of all reads map to introns. Also, we show that these intronic reads are informative about expression levels, significantly increasing the number of detected genes and improving the cluster resolution. zUMIs flexibility makes if possible to accommodate data generated with any of the major scRNA-seq protocols that use BCs and UMIs and is the most feature-rich, fast, and user-friendly pipeline to process such scRNA-seq data.

Sequential Levels of Reading Skills, Prekindergarten--Grade 12.

ERIC Educational Resources Information Center

New York City Board of Education, Brooklyn, NY.

This guide is designed to help teachers, staff members responsible for teacher training, and reading supervisors provide better reading instruction. The skills that lead to mature reading are arranged on eight levels of developmental sequence. Level A is concerned with developing prereading skills. Levels B to D treat initiating and developing…

The Group Reading Inventory in the Social Studies Classroom.

ERIC Educational Resources Information Center

Henk, William A.; Helfeldt, John P.

1985-01-01

The Group Reading Inventory (GRI) is a reading evaluation tool that can survey an entire class of students at the same time. A testing sequence using GRI that allows teachers to identify the functional reading levels of most students in three short examination sessions is presented. (RM)

Using Literature Response Activities To Build Strategic Reading for Students with Reading Difficulties.

ERIC Educational Resources Information Center

Martin, Sarah H.; Martin, Michael A.

2001-01-01

Describes two classroom activities that can be implemented in accordance with the best practices revealed by current research on reading instruction with learning disabled students. Describes what research suggests for promoting comprehension for students with reading difficulties. Describes instructional sequences for two literacy activities,…

Synchronized navigation of current and prior studies using image registration improves radiologist's efficiency.

PubMed

Forsberg, Daniel; Gupta, Amit; Mills, Christopher; MacAdam, Brett; Rosipko, Beverly; Bangert, Barbara A; Coffey, Michael D; Kosmas, Christos; Sunshine, Jeffrey L

2017-03-01

The purpose of this study was to investigate how the use of multi-modal rigid image registration integrated within a standard picture archiving and communication system affects the efficiency of a radiologist while performing routine interpretations of cases including prior examinations. Six radiologists were recruited to read a set of cases (either 16 neuroradiology or 14 musculoskeletal cases) during two crossover reading sessions. Each radiologist read each case twice, one time with synchronized navigation, which enables spatial synchronization across examinations from different study dates, and one time without. Efficiency was evaluated based upon time to read a case and amount of scrolling while browsing a case using Wilcoxon signed rank test. Significant improvements in efficiency were found considering either all radiologists simultaneously, the two sections separately and the majority of individual radiologists for time to read and for amount of scrolling. The relative improvement for each individual radiologist ranged from 4 to 32% for time to read and from 14 to 38% for amount of scrolling. Image registration providing synchronized navigation across examinations from different study dates provides a tool that enables radiologists to work more efficiently while reading cases with one or more prior examinations.

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

PubMed Central

Boccara, Martine; Hamilton, William D. O.; Baulcombe, David C.

1986-01-01

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor. ImagesFig. 2.Fig. 3. PMID:16453668

Allele-specific copy-number discovery from whole-genome and whole-exome sequencing

PubMed Central

Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J.; Szatkiewicz, Jin P.

2015-01-01

Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

MetaRNA-Seq: An Interactive Tool to Browse and Annotate Metadata from RNA-Seq Studies.

PubMed

Kumar, Pankaj; Halama, Anna; Hayat, Shahina; Billing, Anja M; Gupta, Manish; Yousri, Noha A; Smith, Gregory M; Suhre, Karsten

2015-01-01

The number of RNA-Seq studies has grown in recent years. The design of RNA-Seq studies varies from very simple (e.g., two-condition case-control) to very complicated (e.g., time series involving multiple samples at each time point with separate drug treatments). Most of these publically available RNA-Seq studies are deposited in NCBI databases, but their metadata are scattered throughout four different databases: Sequence Read Archive (SRA), Biosample, Bioprojects, and Gene Expression Omnibus (GEO). Although the NCBI web interface is able to provide all of the metadata information, it often requires significant effort to retrieve study- or project-level information by traversing through multiple hyperlinks and going to another page. Moreover, project- and study-level metadata lack manual or automatic curation by categories, such as disease type, time series, case-control, or replicate type, which are vital to comprehending any RNA-Seq study. Here we describe "MetaRNA-Seq," a new tool for interactively browsing, searching, and annotating RNA-Seq metadata with the capability of semiautomatic curation at the study level.

The Mark 3 data base handler

NASA Technical Reports Server (NTRS)

Ryan, J. W.; Ma, C.; Schupler, B. R.

1980-01-01

A data base handler which would act to tie Mark 3 system programs together is discussed. The data base handler is written in FORTRAN and is implemented on the Hewlett-Packard 21MX and the IBM 360/91. The system design objectives were to (1) provide for an easily specified method of data interchange among programs, (2) provide for a high level of data integrity, (3) accommodate changing requirments, (4) promote program accountability, (5) provide a single source of program constants, and (6) provide a central point for data archiving. The system consists of two distinct parts: a set of files existing on disk packs and tapes; and a set of utility subroutines which allow users to access the information in these files. Users never directly read or write the files and need not know the details of how the data are formatted in the files. To the users, the storage medium is format free. A user does need to know something about the sequencing of his data in the files but nothing about data in which he has no interest.

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform, Version 1.5 and 1.x.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chain, Patrick; Lo, Chien-Chi; Li, Po-E

EDGE bioinformatics was developed to help biologists process Next Generation Sequencing data (in the form of raw FASTQ files), even if they have little to no bioinformatics expertise. EDGE is a highly integrated and interactive web-based platform that is capable of running many of the standard analyses that biologists require for viral, bacterial/archaeal, and metagenomic samples. EDGE provides the following analytical workflows: quality trimming and host removal, assembly and annotation, comparisons against known references, taxonomy classification of reads and contigs, whole genome SNP-based phylogenetic analysis, and PCR analysis. EDGE provides an intuitive web-based interface for user input, allows users tomore » visualize and interact with selected results (e.g. JBrowse genome browser), and generates a final detailed PDF report. Results in the form of tables, text files, graphic files, and PDFs can be downloaded. A user management system allows tracking of an individual’s EDGE runs, along with the ability to share, post publicly, delete, or archive their results.« less

Readiness in the Basal Reader: An Update.

ERIC Educational Resources Information Center

Perkins, Pamela

A study examined two 1989 basal reading series' (published by McGraw Hill and Holt) readiness/priming sequences in order to ascertain the theoretical bases of each and then compared the findings with those of an earlier study. All pages of the readiness/priming sequence student texts and workbooks of both basal reading series were analyzed using…

Reference quality assembly of the 3.5 Gb genome of Capsicum annuum form a single linked-read library

USDA-ARS?s Scientific Manuscript database

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Cap...

Reading Nature from a "Bottom-Up" Perspective

ERIC Educational Resources Information Center

Magntorn, Ola; Hellden, Gustav

2007-01-01

This paper reports on a study of ecology teaching and learning in a Swedish primary school class (age 10-11 yrs). A teaching sequence was designed to help students read nature in a river ecosystem. The teaching sequence had a "bottom up" approach, taking as its starting point a common key organism--the freshwater shrimp. From this…

IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

PubMed Central

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

Motivation 16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. Results We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. Availability and Implementation IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq. PMID:25506826

An improved filtering algorithm for big read datasets and its application to single-cell assembly.

PubMed

Wedemeyer, Axel; Kliemann, Lasse; Srivastav, Anand; Schielke, Christian; Reusch, Thorsten B; Rosenstiel, Philip

2017-07-03

For single-cell or metagenomic sequencing projects, it is necessary to sequence with a very high mean coverage in order to make sure that all parts of the sample DNA get covered by the reads produced. This leads to huge datasets with lots of redundant data. A filtering of this data prior to assembly is advisable. Brown et al. (2012) presented the algorithm Diginorm for this purpose, which filters reads based on the abundance of their k-mers. We present Bignorm, a faster and quality-conscious read filtering algorithm. An important new algorithmic feature is the use of phred quality scores together with a detailed analysis of the k-mer counts to decide which reads to keep. We qualify and recommend parameters for our new read filtering algorithm. Guided by these parameters, we remove in terms of median 97.15% of the reads while keeping the mean phred score of the filtered dataset high. Using the SDAdes assembler, we produce assemblies of high quality from these filtered datasets in a fraction of the time needed for an assembly from the datasets filtered with Diginorm. We conclude that read filtering is a practical and efficient method for reducing read data and for speeding up the assembly process. This applies not only for single cell assembly, as shown in this paper, but also to other projects with high mean coverage datasets like metagenomic sequencing projects. Our Bignorm algorithm allows assemblies of competitive quality in comparison to Diginorm, while being much faster. Bignorm is available for download at https://git.informatik.uni-kiel.de/axw/Bignorm .

Accurate estimation of short read mapping quality for next-generation genome sequencing

PubMed Central

Ruffalo, Matthew; Koyutürk, Mehmet; Ray, Soumya; LaFramboise, Thomas

2012-01-01

Motivation: Several software tools specialize in the alignment of short next-generation sequencing reads to a reference sequence. Some of these tools report a mapping quality score for each alignment—in principle, this quality score tells researchers the likelihood that the alignment is correct. However, the reported mapping quality often correlates weakly with actual accuracy and the qualities of many mappings are underestimated, encouraging the researchers to discard correct mappings. Further, these low-quality mappings tend to correlate with variations in the genome (both single nucleotide and structural), and such mappings are important in accurately identifying genomic variants. Approach: We develop a machine learning tool, LoQuM (LOgistic regression tool for calibrating the Quality of short read mappings, to assign reliable mapping quality scores to mappings of Illumina reads returned by any alignment tool. LoQuM uses statistics on the read (base quality scores reported by the sequencer) and the alignment (number of matches, mismatches and deletions, mapping quality score returned by the alignment tool, if available, and number of mappings) as features for classification and uses simulated reads to learn a logistic regression model that relates these features to actual mapping quality. Results: We test the predictions of LoQuM on an independent dataset generated by the ART short read simulation software and observe that LoQuM can ‘resurrect’ many mappings that are assigned zero quality scores by the alignment tools and are therefore likely to be discarded by researchers. We also observe that the recalibration of mapping quality scores greatly enhances the precision of called single nucleotide polymorphisms. Availability: LoQuM is available as open source at http://compbio.case.edu/loqum/. Contact: matthew.ruffalo@case.edu. PMID:22962451

RIEMS: a software pipeline for sensitive and comprehensive taxonomic classification of reads from metagenomics datasets.

PubMed

Scheuch, Matthias; Höper, Dirk; Beer, Martin

2015-03-03

Fuelled by the advent and subsequent development of next generation sequencing technologies, metagenomics became a powerful tool for the analysis of microbial communities both scientifically and diagnostically. The biggest challenge is the extraction of relevant information from the huge sequence datasets generated for metagenomics studies. Although a plethora of tools are available, data analysis is still a bottleneck. To overcome the bottleneck of data analysis, we developed an automated computational workflow called RIEMS - Reliable Information Extraction from Metagenomic Sequence datasets. RIEMS assigns every individual read sequence within a dataset taxonomically by cascading different sequence analyses with decreasing stringency of the assignments using various software applications. After completion of the analyses, the results are summarised in a clearly structured result protocol organised taxonomically. The high accuracy and performance of RIEMS analyses were proven in comparison with other tools for metagenomics data analysis using simulated sequencing read datasets. RIEMS has the potential to fill the gap that still exists with regard to data analysis for metagenomics studies. The usefulness and power of RIEMS for the analysis of genuine sequencing datasets was demonstrated with an early version of RIEMS in 2011 when it was used to detect the orthobunyavirus sequences leading to the discovery of Schmallenberg virus.

Rapid Threat Organism Recognition Pipeline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Kelly P.; Solberg, Owen D.; Schoeniger, Joseph S.

2013-05-07

The RAPTOR computational pipeline identifies microbial nucleic acid sequences present in sequence data from clinical samples. It takes as input raw short-read genomic sequence data (in particular, the type generated by the Illumina sequencing platforms) and outputs taxonomic evaluation of detected microbes in various human-readable formats. This software was designed to assist in the diagnosis or characterization of infectious disease, by detecting pathogen sequences in nucleic acid sequence data from clinical samples. It has also been applied in the detection of algal pathogens, when algal biofuel ponds became unproductive. RAPTOR first trims and filters genomic sequence reads based on qualitymore » and related considerations, then performs a quick alignment to the human (or other host) genome to filter out host sequences, then performs a deeper search against microbial genomes. Alignment to a protein sequence database is optional. Alignment results are summarized and placed in a taxonomic framework using the Lowest Common Ancestor algorithm.« less

GRIM-Filter: Fast seed location filtering in DNA read mapping using processing-in-memory technologies.

PubMed

Kim, Jeremie S; Senol Cali, Damla; Xin, Hongyi; Lee, Donghyuk; Ghose, Saugata; Alser, Mohammed; Hassan, Hasan; Ergin, Oguz; Alkan, Can; Mutlu, Onur

2018-05-09

Seed location filtering is critical in DNA read mapping, a process where billions of DNA fragments (reads) sampled from a donor are mapped onto a reference genome to identify genomic variants of the donor. State-of-the-art read mappers 1) quickly generate possible mapping locations for seeds (i.e., smaller segments) within each read, 2) extract reference sequences at each of the mapping locations, and 3) check similarity between each read and its associated reference sequences with a computationally-expensive algorithm (i.e., sequence alignment) to determine the origin of the read. A seed location filter comes into play before alignment, discarding seed locations that alignment would deem a poor match. The ideal seed location filter would discard all poor match locations prior to alignment such that there is no wasted computation on unnecessary alignments. We propose a novel seed location filtering algorithm, GRIM-Filter, optimized to exploit 3D-stacked memory systems that integrate computation within a logic layer stacked under memory layers, to perform processing-in-memory (PIM). GRIM-Filter quickly filters seed locations by 1) introducing a new representation of coarse-grained segments of the reference genome, and 2) using massively-parallel in-memory operations to identify read presence within each coarse-grained segment. Our evaluations show that for a sequence alignment error tolerance of 0.05, GRIM-Filter 1) reduces the false negative rate of filtering by 5.59x-6.41x, and 2) provides an end-to-end read mapper speedup of 1.81x-3.65x, compared to a state-of-the-art read mapper employing the best previous seed location filtering algorithm. GRIM-Filter exploits 3D-stacked memory, which enables the efficient use of processing-in-memory, to overcome the memory bandwidth bottleneck in seed location filtering. We show that GRIM-Filter significantly improves the performance of a state-of-the-art read mapper. GRIM-Filter is a universal seed location filter that can be applied to any read mapper. We hope that our results provide inspiration for new works to design other bioinformatics algorithms that take advantage of emerging technologies and new processing paradigms, such as processing-in-memory using 3D-stacked memory devices.

Arioc: high-throughput read alignment with GPU-accelerated exploration of the seed-and-extend search space

PubMed Central

Budavari, Tamas; Langmead, Ben; Wheelan, Sarah J.; Salzberg, Steven L.; Szalay, Alexander S.

2015-01-01

When computing alignments of DNA sequences to a large genome, a key element in achieving high processing throughput is to prioritize locations in the genome where high-scoring mappings might be expected. We formulated this task as a series of list-processing operations that can be efficiently performed on graphics processing unit (GPU) hardware.We followed this approach in implementing a read aligner called Arioc that uses GPU-based parallel sort and reduction techniques to identify high-priority locations where potential alignments may be found. We then carried out a read-by-read comparison of Arioc’s reported alignments with the alignments found by several leading read aligners. With simulated reads, Arioc has comparable or better accuracy than the other read aligners we tested. With human sequencing reads, Arioc demonstrates significantly greater throughput than the other aligners we evaluated across a wide range of sensitivity settings. The Arioc software is available at https://github.com/RWilton/Arioc. It is released under a BSD open-source license. PMID:25780763

Efficient radiologic reading environment by using an open-source macro program as connection software.

PubMed

Lee, Young Han

2012-01-01

The objectives are (1) to introduce an easy open-source macro program as connection software and (2) to illustrate the practical usages in radiologic reading environment by simulating the radiologic reading process. The simulation is a set of radiologic reading process to do a practical task in the radiologic reading room. The principal processes are: (1) to view radiologic images on the Picture Archiving and Communicating System (PACS), (2) to connect the HIS/EMR (Hospital Information System/Electronic Medical Record) system, (3) to make an automatic radiologic reporting system, and (4) to record and recall information of interesting cases. This simulation environment was designed by using open-source macro program as connection software. The simulation performed well on the Window-based PACS workstation. Radiologists practiced the steps of the simulation comfortably by utilizing the macro-powered radiologic environment. This macro program could automate several manual cumbersome steps in the radiologic reading process. This program successfully acts as connection software for the PACS software, EMR/HIS, spreadsheet, and other various input devices in the radiologic reading environment. A user-friendly efficient radiologic reading environment could be established by utilizing open-source macro program as connection software. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

PubMed Central

Karamitros, Timokratis; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal. PMID:27309375

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.

PubMed

Karamitros, Timokratis; Harrison, Ian; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.

HIA: a genome mapper using hybrid index-based sequence alignment.

PubMed

Choi, Jongpill; Park, Kiejung; Cho, Seong Beom; Chung, Myungguen

2015-01-01

A number of alignment tools have been developed to align sequencing reads to the human reference genome. The scale of information from next-generation sequencing (NGS) experiments, however, is increasing rapidly. Recent studies based on NGS technology have routinely produced exome or whole-genome sequences from several hundreds or thousands of samples. To accommodate the increasing need of analyzing very large NGS data sets, it is necessary to develop faster, more sensitive and accurate mapping tools. HIA uses two indices, a hash table index and a suffix array index. The hash table performs direct lookup of a q-gram, and the suffix array performs very fast lookup of variable-length strings by exploiting binary search. We observed that combining hash table and suffix array (hybrid index) is much faster than the suffix array method for finding a substring in the reference sequence. Here, we defined the matching region (MR) is a longest common substring between a reference and a read. And, we also defined the candidate alignment regions (CARs) as a list of MRs that is close to each other. The hybrid index is used to find candidate alignment regions (CARs) between a reference and a read. We found that aligning only the unmatched regions in the CAR is much faster than aligning the whole CAR. In benchmark analysis, HIA outperformed in mapping speed compared with the other aligners, without significant loss of mapping accuracy. Our experiments show that the hybrid of hash table and suffix array is useful in terms of speed for mapping NGS sequencing reads to the human reference genome sequence. In conclusion, our tool is appropriate for aligning massive data sets generated by NGS sequencing.

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease.

PubMed

Dilliott, Allison A; Farhan, Sali M K; Ghani, Mahdi; Sato, Christine; Liang, Eric; Zhang, Ming; McIntyre, Adam D; Cao, Henian; Racacho, Lemuel; Robinson, John F; Strong, Michael J; Masellis, Mario; Bulman, Dennis E; Rogaeva, Ekaterina; Lang, Anthony; Tartaglia, Carmela; Finger, Elizabeth; Zinman, Lorne; Turnbull, John; Freedman, Morris; Swartz, Rick; Black, Sandra E; Hegele, Robert A

2018-04-04

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PubMed

Straub, Shannon C K; Parks, Matthew; Weitemier, Kevin; Fishbein, Mark; Cronn, Richard C; Liston, Aaron

2012-02-01

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

BOOK REVIEW: Treasure-Hunting in Astronomical Plate Archives.

NASA Astrophysics Data System (ADS)

Kroll, Peter; La Dous, Constanze; Brauer, Hans-Juergen; Sterken, C.

This book consists of the proceedings of a conference on the exploration of the invaluable scientific treasure present in astronomical plate archives worldwide. The book incorporates fifty scientific papers covering almost 250 pages. There are several most useful papers, such as, for example, an introduction to the world's large plate archives that serves the purpose of a guide for the beginning user of plate archives. It includes a very useful list of twelve mayor archives with many details on their advantages (completeness, number of plates, classification system and homogeneity of time coverage) and their limitations (plate quality, access, electronic catalogues, photographic services, limiting magnitudes, search software and cost to the user). Other topics cover available contemporary digitization machines, the applications of commercial flatbed scanners, technical aspects of plate consulting, astrophysical applications and astrometric uses, data reduction, data archiving and retrieval, and strategies to find astrophysically useful information on plates. The astrophysical coverage is very broad: from solar-system bodies to variable stars, sky surveys and sky patrols covering the galactic and extragalactic domain and even gravitational lensing. The book concludes by an illuminating paper on ALADIN, the reference tool for identification of astronomical sources. This work can be considered as a kind of field guide, and is recommended reading for anyone who wishes to undertake small- or large-scale consulting of photographic plate material. A shortcoming of the proceedings is the fact that very few papers have abstracts. BOOK REVIEW: Treasure-Hunting in Astronomical Plate Archives. Proceedings of the international workshop held at Sonneberg Observatory, March 4-6, 1999. Peter Kroll, Constanze la Dous and Hans-Juergen Brauer (Eds.)

The present and future of de novo whole-genome assembly.

PubMed

Sohn, Jang-Il; Nam, Jin-Wu

2018-01-01

As the advent of next-generation sequencing (NGS) technology, various de novo assembly algorithms based on the de Bruijn graph have been developed to construct chromosome-level sequences. However, numerous technical or computational challenges in de novo assembly still remain, although many bright ideas and heuristics have been suggested to tackle the challenges in both experimental and computational settings. In this review, we categorize de novo assemblers on the basis of the type of de Bruijn graphs (Hamiltonian and Eulerian) and discuss the challenges of de novo assembly for short NGS reads regarding computational complexity and assembly ambiguity. Then, we discuss how the limitations of the short reads can be overcome by using a single-molecule sequencing platform that generates long reads of up to several kilobases. In fact, the long read assembly has caused a paradigm shift in whole-genome assembly in terms of algorithms and supporting steps. We also summarize (i) hybrid assemblies using both short and long reads and (ii) overlap-based assemblies for long reads and discuss their challenges and future prospects. This review provides guidelines to determine the optimal approach for a given input data type, computational budget or genome. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes.

PubMed

Fredlake, Christopher P; Hert, Daniel G; Kan, Cheuk-Wai; Chiesl, Thomas N; Root, Brian E; Forster, Ryan E; Barron, Annelise E

2008-01-15

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

PubMed Central

Fredlake, Christopher P.; Hert, Daniel G.; Kan, Cheuk-Wai; Chiesl, Thomas N.; Root, Brian E.; Forster, Ryan E.; Barron, Annelise E.

2008-01-01

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require ≈70 min to deliver ≈650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered “hybrid” mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs. PMID:18184818

Implementing Replacement Cost Accounting

DTIC Science & Technology

1976-12-01

cost accounting Clickener, John Ross Monterey, California. Naval Postgraduate School http://hdl.handle.net/10945/17810 Downloaded from NPS Archive...Calhoun IMPLEMENTING REPLACEMENT COST ACCOUNTING John Ross CHckener NAVAL POSTGRADUATE SCHOOL Monterey, California THESIS IMPLEMENTING REPLACEMENT COST ...Implementing Replacement Cost Accounting 7. AUTHORS John Ross Clickener READ INSTRUCTIONS BEFORE COMPLETING FORM 3. RECIPIENT’S CATALOG NUMBER 9. TYRE OF

Academic Success of At-Risk African American Male Students Who Receive Culturally Relevant Teaching, College Readiness Preparation, and Mentorship

ERIC Educational Resources Information Center

Taylor, Timothy L.

2016-01-01

This quantitative study analyzed archival data to determine whether a significant difference existed in the reading comprehension scores and student success (enrollment in honors and or advanced placement classes and college after graduation) of at-risk African American male students who received Advancement via Individual Determination/African…

Archives of Environmental Health, Volume 18 Number 4. Ninth AMA Air Pollution Medical Research Conference, Denver, July 22-24, 1968.

ERIC Educational Resources Information Center

Barton, Frank

Papers read before the Ninth American Medical Association (AMA) Air Pollution Medical Research Conference, Denver, Colorado, July 22-24, 1968, are presented in this document. Topics deal with the relationship and effects of atmospheric pollution to respiratory diseases, epidemiology, human physiological reactions, urban morbidity, health of school…

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

PubMed Central

2011-01-01

Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. Conclusion An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml). PMID:21266061

MutAid: Sanger and NGS Based Integrated Pipeline for Mutation Identification, Validation and Annotation in Human Molecular Genetics.

PubMed

Pandey, Ram Vinay; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a new pipeline called MutAid to analyze and interpret raw sequencing data produced by Sanger or several NGS sequencing platforms. It performs format conversion, base calling, quality trimming, filtering, read mapping, variant calling, variant annotation and analysis of Sanger and NGS data under a single platform. It is capable of analyzing reads from multiple patients in a single run to create a list of potential disease causing base substitutions as well as insertions and deletions. MutAid has been developed for expert and non-expert users and supports four sequencing platforms including Sanger, Illumina, 454 and Ion Torrent. Furthermore, for NGS data analysis, five read mappers including BWA, TMAP, Bowtie, Bowtie2 and GSNAP and four variant callers including GATK-HaplotypeCaller, SAMTOOLS, Freebayes and VarScan2 pipelines are supported. MutAid is freely available at https://sourceforge.net/projects/mutaid.

MutAid: Sanger and NGS Based Integrated Pipeline for Mutation Identification, Validation and Annotation in Human Molecular Genetics

PubMed Central

Pandey, Ram Vinay; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

2016-01-01

Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a new pipeline called MutAid to analyze and interpret raw sequencing data produced by Sanger or several NGS sequencing platforms. It performs format conversion, base calling, quality trimming, filtering, read mapping, variant calling, variant annotation and analysis of Sanger and NGS data under a single platform. It is capable of analyzing reads from multiple patients in a single run to create a list of potential disease causing base substitutions as well as insertions and deletions. MutAid has been developed for expert and non-expert users and supports four sequencing platforms including Sanger, Illumina, 454 and Ion Torrent. Furthermore, for NGS data analysis, five read mappers including BWA, TMAP, Bowtie, Bowtie2 and GSNAP and four variant callers including GATK-HaplotypeCaller, SAMTOOLS, Freebayes and VarScan2 pipelines are supported. MutAid is freely available at https://sourceforge.net/projects/mutaid. PMID:26840129

The Effects of CBI Lesson Sequence Type and Field Dependence on Learning from Computer-Based Cooperative Instruction in Web

ERIC Educational Resources Information Center

Ipek, Ismail

2010-01-01

The purpose of this study was to investigate the effects of CBI lesson sequence type and cognitive style of field dependence on learning from Computer-Based Cooperative Instruction (CBCI) in WEB on the dependent measures, achievement, reading comprehension and reading rate. Eighty-seven college undergraduate students were randomly assigned to…

Short-read, high-throughput sequencing technology for STR genotyping

PubMed Central

Bornman, Daniel M.; Hester, Mark E.; Schuetter, Jared M.; Kasoji, Manjula D.; Minard-Smith, Angela; Barden, Curt A.; Nelson, Scott C.; Godbold, Gene D.; Baker, Christine H.; Yang, Boyu; Walther, Jacquelyn E.; Tornes, Ivan E.; Yan, Pearlly S.; Rodriguez, Benjamin; Bundschuh, Ralf; Dickens, Michael L.; Young, Brian A.; Faith, Seth A.

2013-01-01

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples. PMID:25621315

Accurate Typing of Human Leukocyte Antigen Class I Genes by Oxford Nanopore Sequencing.

PubMed

Liu, Chang; Xiao, Fangzhou; Hoisington-Lopez, Jessica; Lang, Kathrin; Quenzel, Philipp; Duffy, Brian; Mitra, Robi David

2018-04-03

Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunologic compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. We developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent data sets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the two-field resolution. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Identifying micro-inversions using high-throughput sequencing reads.

PubMed

He, Feifei; Li, Yang; Tang, Yu-Hang; Ma, Jian; Zhu, Huaiqiu

2016-01-11

The identification of inversions of DNA segments shorter than read length (e.g., 100 bp), defined as micro-inversions (MIs), remains challenging for next-generation sequencing reads. It is acknowledged that MIs are important genomic variation and may play roles in causing genetic disease. However, current alignment methods are generally insensitive to detect MIs. Here we develop a novel tool, MID (Micro-Inversion Detector), to identify MIs in human genomes using next-generation sequencing reads. The algorithm of MID is designed based on a dynamic programming path-finding approach. What makes MID different from other variant detection tools is that MID can handle small MIs and multiple breakpoints within an unmapped read. Moreover, MID improves reliability in low coverage data by integrating multiple samples. Our evaluation demonstrated that MID outperforms Gustaf, which can currently detect inversions from 30 bp to 500 bp. To our knowledge, MID is the first method that can efficiently and reliably identify MIs from unmapped short next-generation sequencing reads. MID is reliable on low coverage data, which is suitable for large-scale projects such as the 1000 Genomes Project (1KGP). MID identified previously unknown MIs from the 1KGP that overlap with genes and regulatory elements in the human genome. We also identified MIs in cancer cell lines from Cancer Cell Line Encyclopedia (CCLE). Therefore our tool is expected to be useful to improve the study of MIs as a type of genetic variant in the human genome. The source code can be downloaded from: http://cqb.pku.edu.cn/ZhuLab/MID .

A parallel and sensitive software tool for methylation analysis on multicore platforms.

PubMed

Tárraga, Joaquín; Pérez, Mariano; Orduña, Juan M; Duato, José; Medina, Ignacio; Dopazo, Joaquín

2015-10-01

DNA methylation analysis suffers from very long processing time, as the advent of Next-Generation Sequencers has shifted the bottleneck of genomic studies from the sequencers that obtain the DNA samples to the software that performs the analysis of these samples. The existing software for methylation analysis does not seem to scale efficiently neither with the size of the dataset nor with the length of the reads to be analyzed. As it is expected that the sequencers will provide longer and longer reads in the near future, efficient and scalable methylation software should be developed. We present a new software tool, called HPG-Methyl, which efficiently maps bisulphite sequencing reads on DNA, analyzing DNA methylation. The strategy used by this software consists of leveraging the speed of the Burrows-Wheeler Transform to map a large number of DNA fragments (reads) rapidly, as well as the accuracy of the Smith-Waterman algorithm, which is exclusively employed to deal with the most ambiguous and shortest reads. Experimental results on platforms with Intel multicore processors show that HPG-Methyl significantly outperforms in both execution time and sensitivity state-of-the-art software such as Bismark, BS-Seeker or BSMAP, particularly for long bisulphite reads. Software in the form of C libraries and functions, together with instructions to compile and execute this software. Available by sftp to anonymous@clariano.uv.es (password 'anonymous'). juan.orduna@uv.es or jdopazo@cipf.es. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes

PubMed Central

Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

2016-01-01

The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter.’ We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about the presence and prevalence of giant viruses in the environment and the human body. PMID:27065984

Possible roles for fronto-striatal circuits in reading disorder

PubMed Central

Hancock, Roeland; Richlan, Fabio; Hoeft, Fumiko

2016-01-01

Several studies have reported hyperactivation in frontal and striatal regions in individuals with reading disorder (RD) during reading-related tasks. Hyperactivation in these regions is typically interpreted as a form of neural compensation and related to articulatory processing. Fronto-striatal hyperactivation in RD can however, also arise from fundamental impairment in reading related processes, such as phonological processing and implicit sequence learning relevant to early language acquisition. We review current evidence for the compensation hypothesis in RD and apply large-scale reverse inference to investigate anatomical overlap between hyperactivation regions and neural systems for articulation, phonological processing, implicit sequence learning. We found anatomical convergence between hyperactivation regions and regions supporting articulation, consistent with the proposed compensatory role of these regions, and low convergence with phonological and implicit sequence learning regions. Although the application of large-scale reverse inference to decode function in a clinical population should be interpreted cautiously, our findings suggest future lines of research that may clarify the functional significance of hyperactivation in RD. PMID:27826071

The advantages of SMRT sequencing.

PubMed

Roberts, Richard J; Carneiro, Mauricio O; Schatz, Michael C

2013-07-03

Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful technology and an ideal approach to the complete sequencing of small genomes.

Metagenomic ventures into outer sequence space.

PubMed

Dutilh, Bas E

Sequencing DNA or RNA directly from the environment often results in many sequencing reads that have no homologs in the database. These are referred to as "unknowns," and reflect the vast unexplored microbial sequence space of our biosphere, also known as "biological dark matter." However, unknowns also exist because metagenomic datasets are not optimally mined. There is a pressure on researchers to publish and move on, and the unknown sequences are often left for what they are, and conclusions drawn based on reads with annotated homologs. This can cause abundant and widespread genomes to be overlooked, such as the recently discovered human gut bacteriophage crAssphage. The unknowns may be enriched for bacteriophage sequences, the most abundant and genetically diverse component of the biosphere and of sequence space. However, it remains an open question, what is the actual size of biological sequence space? The de novo assembly of shotgun metagenomes is the most powerful tool to address this question.

An outbreak of respiratory tularemia caused by diverse clones of Francisella tularensis.

PubMed

Johansson, Anders; Lärkeryd, Adrian; Widerström, Micael; Mörtberg, Sara; Myrtännäs, Kerstin; Ohrman, Caroline; Birdsell, Dawn; Keim, Paul; Wagner, David M; Forsman, Mats; Larsson, Pär

2014-12-01

The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1-3 of 1 585 229 examined nucleotides. The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

PubMed

Hong, Lewis Z; Hong, Shuzhen; Wong, Han Teng; Aw, Pauline P K; Cheng, Yan; Wilm, Andreas; de Sessions, Paola F; Lim, Seng Gee; Nagarajan, Niranjan; Hibberd, Martin L; Quake, Stephen R; Burkholder, William F

2014-01-01

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

XS: a FASTQ read simulator.

PubMed

Pratas, Diogo; Pinho, Armando J; Rodrigues, João M O S

2014-01-16

The emerging next-generation sequencing (NGS) is bringing, besides the natural huge amounts of data, an avalanche of new specialized tools (for analysis, compression, alignment, among others) and large public and private network infrastructures. Therefore, a direct necessity of specific simulation tools for testing and benchmarking is rising, such as a flexible and portable FASTQ read simulator, without the need of a reference sequence, yet correctly prepared for producing approximately the same characteristics as real data. We present XS, a skilled FASTQ read simulation tool, flexible, portable (does not need a reference sequence) and tunable in terms of sequence complexity. It has several running modes, depending on the time and memory available, and is aimed at testing computing infrastructures, namely cloud computing of large-scale projects, and testing FASTQ compression algorithms. Moreover, XS offers the possibility of simulating the three main FASTQ components individually (headers, DNA sequences and quality-scores). XS provides an efficient and convenient method for fast simulation of FASTQ files, such as those from Ion Torrent (currently uncovered by other simulators), Roche-454, Illumina and ABI-SOLiD sequencing machines. This tool is publicly available at http://bioinformatics.ua.pt/software/xs/.

Stress Sensitivity and Reading Performance in Spanish: A Study with Children

ERIC Educational Resources Information Center

Gutierrez-Palma, Nicolas; Reyes, Alfonso Palma

2007-01-01

This paper investigates the relationship between ability to detect changes in prosody and reading performance in Spanish. Participants were children aged 7-8 years. Their tasks consisted of reading words, reading non-words, stressing non-words and reproducing sequences of two, three or four non-words by pressing the corresponding keys on the…

Closet to Cloud: The online archiving of tape-based continuous NCSN seismic data from 1993-2005

NASA Astrophysics Data System (ADS)

Neuhauser, D. S.; Aranha, M. A.; Kohler, W. M.; Oppenheimer, D.

2016-12-01

As earthquake monitoring systems in the 1980s moved from analog to digital recording systems, most seismic networks only archived digital waveforms from detected events due to lack of affordable online digital storage for continuous high-rate (100 sps) data. The Northern California Earthquake Data Center (NCEDC), established in 1991 by UC Berkeley and the USGS Menlo Park, archived 20 sps continuous data and triggerd high-rate from the sparse Berkeley seismic network, but could not afford the online storage for continuous high-rate data from the 300+ stations of the USGS Northern California Seismic Network (NCSN). The discovery of non-volcanic tremor and the use of continuous waveform correlation techniques for detecting repeating earthquakes combined with the increase in disk capacity capacity and significant reduction in disk costs led the Northern California Earthquake Data Center (NCEDC) to begin archiving continuous high-rate waveforms in 2004-2005. The USGS Menlo Park NCSN network had backup tapes of continuous high-rate waveform data since 1993 on the shelf, and the USGS and NCEDC embarked on a project to restore and archive all continuous NCSN data from 1993 through 2005. We will discuss the procedures and problems encountered when reading, transcribing, converting data formats, SEED channel naming, and archiving the 1993-2005 continuous NCSN waveforms. We will also illustrate new science enabled by these data. These and other northern California seismic and geophysical data are available via web services at http://service.ncedc.org

Use of the Minion nanopore sequencer for rapid sequencing of avian influenza virus isolates

USDA-ARS?s Scientific Manuscript database

A relatively new sequencing technology, the MinION nanopore sequencer, provides a platform that is smaller, faster, and cheaper than existing Next Generation Sequence (NGS) technologies. The MinION sequences of individual strands of DNA and can produce millions of sequencing reads. The cost of the s...

The long reads ahead: de novo genome assembly using the MinION

PubMed Central

de Lannoy, Carlos; de Ridder, Dick; Risse, Judith

2017-01-01

Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of overview of the expanding landscape of analysis tools, as performance evaluations are outdated quickly. As the MinION is approaching a state of maturity, its user community would benefit from a thorough comparative benchmarking effort of de novo assembly pipelines in the near future. An earlier version of this article can be found on  bioRxiv. PMID:29375809

Accurate read-based metagenome characterization using a hierarchical suite of unique signatures

PubMed Central

Freitas, Tracey Allen K.; Li, Po-E; Scholz, Matthew B.; Chain, Patrick S. G.

2015-01-01

A major challenge in the field of shotgun metagenomics is the accurate identification of organisms present within a microbial community, based on classification of short sequence reads. Though existing microbial community profiling methods have attempted to rapidly classify the millions of reads output from modern sequencers, the combination of incomplete databases, similarity among otherwise divergent genomes, errors and biases in sequencing technologies, and the large volumes of sequencing data required for metagenome sequencing has led to unacceptably high false discovery rates (FDR). Here, we present the application of a novel, gene-independent and signature-based metagenomic taxonomic profiling method with significantly and consistently smaller FDR than any other available method. Our algorithm circumvents false positives using a series of non-redundant signature databases and examines Genomic Origins Through Taxonomic CHAllenge (GOTTCHA). GOTTCHA was tested and validated on 20 synthetic and mock datasets ranging in community composition and complexity, was applied successfully to data generated from spiked environmental and clinical samples, and robustly demonstrates superior performance compared with other available tools. PMID:25765641

Machine Learned Replacement of N-Labels for Basecalled Sequences in DNA Barcoding.

PubMed

Ma, Eddie Y T; Ratnasingham, Sujeevan; Kremer, Stefan C

2018-01-01

This study presents a machine learning method that increases the number of identified bases in Sanger Sequencing. The system post-processes a KB basecalled chromatogram. It selects a recoverable subset of N-labels in the KB-called chromatogram to replace with basecalls (A,C,G,T). An N-label correction is defined given an additional read of the same sequence, and a human finished sequence. Corrections are added to the dataset when an alignment determines the additional read and human agree on the identity of the N-label. KB must also rate the replacement with quality value of in the additional read. Corrections are only available during system training. Developing the system, nearly 850,000 N-labels are obtained from Barcode of Life Datasystems, the premier database of genetic markers called DNA Barcodes. Increasing the number of correct bases improves reference sequence reliability, increases sequence identification accuracy, and assures analysis correctness. Keeping with barcoding standards, our system maintains an error rate of percent. Our system only applies corrections when it estimates low rate of error. Tested on this data, our automation selects and recovers: 79 percent of N-labels from COI (animal barcode); 80 percent from matK and rbcL (plant barcodes); and 58 percent from non-protein-coding sequences (across eukaryotes).

Systematic evaluation of the impact of ChIP-seq read designs on genome coverage, peak identification, and allele-specific binding detection.

PubMed

Zhang, Qi; Zeng, Xin; Younkin, Sam; Kawli, Trupti; Snyder, Michael P; Keleş, Sündüz

2016-02-24

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments revolutionized genome-wide profiling of transcription factors and histone modifications. Although maturing sequencing technologies allow these experiments to be carried out with short (36-50 bps), long (75-100 bps), single-end, or paired-end reads, the impact of these read parameters on the downstream data analysis are not well understood. In this paper, we evaluate the effects of different read parameters on genome sequence alignment, coverage of different classes of genomic features, peak identification, and allele-specific binding detection. We generated 101 bps paired-end ChIP-seq data for many transcription factors from human GM12878 and MCF7 cell lines. Systematic evaluations using in silico variations of these data as well as fully simulated data, revealed complex interplay between the sequencing parameters and analysis tools, and indicated clear advantages of paired-end designs in several aspects such as alignment accuracy, peak resolution, and most notably, allele-specific binding detection. Our work elucidates the effect of design on the downstream analysis and provides insights to investigators in deciding sequencing parameters in ChIP-seq experiments. We present the first systematic evaluation of the impact of ChIP-seq designs on allele-specific binding detection and highlights the power of pair-end designs in such studies.

Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

PubMed

Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

2015-08-18

Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing

PubMed Central

Vembar, Shruthi Sridhar; Seetin, Matthew; Lambert, Christine; Nattestad, Maria; Schatz, Michael C.; Baybayan, Primo; Scherf, Artur; Smith, Melissa Laird

2016-01-01

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [∼90–99% (A + T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission. PMID:27345719

Association analysis using next-generation sequence data from publicly available control groups: the robust variance score statistic

PubMed Central

Derkach, Andriy; Chiang, Theodore; Gong, Jiafen; Addis, Laura; Dobbins, Sara; Tomlinson, Ian; Houlston, Richard; Pal, Deb K.; Strug, Lisa J.

2014-01-01

Motivation: Sufficiently powered case–control studies with next-generation sequence (NGS) data remain prohibitively expensive for many investigators. If feasible, a more efficient strategy would be to include publicly available sequenced controls. However, these studies can be confounded by differences in sequencing platform; alignment, single nucleotide polymorphism and variant calling algorithms; read depth; and selection thresholds. Assuming one can match cases and controls on the basis of ethnicity and other potential confounding factors, and one has access to the aligned reads in both groups, we investigate the effect of systematic differences in read depth and selection threshold when comparing allele frequencies between cases and controls. We propose a novel likelihood-based method, the robust variance score (RVS), that substitutes genotype calls by their expected values given observed sequence data. Results: We show theoretically that the RVS eliminates read depth bias in the estimation of minor allele frequency. We also demonstrate that, using simulated and real NGS data, the RVS method controls Type I error and has comparable power to the ‘gold standard’ analysis with the true underlying genotypes for both common and rare variants. Availability and implementation: An RVS R script and instructions can be found at strug.research.sickkids.ca, and at https://github.com/strug-lab/RVS. Contact: lisa.strug@utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24733292

BM-Map: Bayesian Mapping of Multireads for Next-Generation Sequencing Data

PubMed Central

Ji, Yuan; Xu, Yanxun; Zhang, Qiong; Tsui, Kam-Wah; Yuan, Yuan; Norris, Clift; Liang, Shoudan; Liang, Han

2011-01-01

Summary Next-generation sequencing (NGS) technology generates millions of short reads, which provide valuable information for various aspects of cellular activities and biological functions. A key step in NGS applications (e.g., RNA-Seq) is to map short reads to correct genomic locations within the source genome. While most reads are mapped to a unique location, a significant proportion of reads align to multiple genomic locations with equal or similar numbers of mismatches; these are called multireads. The ambiguity in mapping the multireads may lead to bias in downstream analyses. Currently, most practitioners discard the multireads in their analysis, resulting in a loss of valuable information, especially for the genes with similar sequences. To refine the read mapping, we develop a Bayesian model that computes the posterior probability of mapping a multiread to each competing location. The probabilities are used for downstream analyses, such as the quantification of gene expression. We show through simulation studies and RNA-Seq analysis of real life data that the Bayesian method yields better mapping than the current leading methods. We provide a C++ program for downloading that is being packaged into a user-friendly software. PMID:21517792

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

Specificity control for read alignments using an artificial reference genome-guided false discovery rate.

PubMed

Giese, Sven H; Zickmann, Franziska; Renard, Bernhard Y

2014-01-01

Accurate estimation, comparison and evaluation of read mapping error rates is a crucial step in the processing of next-generation sequencing data, as further analysis steps and interpretation assume the correctness of the mapping results. Current approaches are either focused on sensitivity estimation and thereby disregard specificity or are based on read simulations. Although continuously improving, read simulations are still prone to introduce a bias into the mapping error quantitation and cannot capture all characteristics of an individual dataset. We introduce ARDEN (artificial reference driven estimation of false positives in next-generation sequencing data), a novel benchmark method that estimates error rates of read mappers based on real experimental reads, using an additionally generated artificial reference genome. It allows a dataset-specific computation of error rates and the construction of a receiver operating characteristic curve. Thereby, it can be used for optimization of parameters for read mappers, selection of read mappers for a specific problem or for filtering alignments based on quality estimation. The use of ARDEN is demonstrated in a general read mapper comparison, a parameter optimization for one read mapper and an application example in single-nucleotide polymorphism discovery with a significant reduction in the number of false positive identifications. The ARDEN source code is freely available at http://sourceforge.net/projects/arden/.

RNA-Seq for Bacterial Gene Expression.

PubMed

Poulsen, Line Dahl; Vinther, Jeppe

2018-06-01

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

PubMed

Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

2009-06-01

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

PubMed

Yohda, Masafumi; Yagi, Osami; Takechi, Ayane; Kitajima, Mizuki; Matsuda, Hisashi; Miyamura, Naoaki; Aizawa, Tomoko; Nakajima, Mutsuyasu; Sunairi, Michio; Daiba, Akito; Miyajima, Takashi; Teruya, Morimi; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Nakano, Kazuma; Aoyama, Misako; Terabayashi, Yasunobu; Satou, Kazuhito; Hirano, Takashi

2015-07-01

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

PubMed

Nielsen, E E; Morgan, J A T; Maher, S L; Edson, J; Gauthier, M; Pepperell, J; Holmes, B J; Bennett, M B; Ovenden, J R

2017-05-01

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield. © 2016 John Wiley & Sons Ltd.

Analyzing Saturn's Magnetospheric Data After Cassini - Improving and Future-Proofing Cassini / MAPS Tools and Data

NASA Astrophysics Data System (ADS)

Brown, L. E.; Faden, J.; Vandegriff, J. D.; Kurth, W. S.; Mitchell, D. G.

2017-12-01

We present a plan to provide enhanced longevity to analysis software and science data used throughout the Cassini mission for viewing Magnetosphere and Plasma Science (MAPS) data. While a final archive is being prepared for Cassini, the tools that read from this archive will eventually become moribund as real world hardware and software systems evolve. We will add an access layer over existing and planned Cassini data products that will allow multiple tools to access many public MAPS datasets. The access layer is called the Heliophysics Application Programmer's Interface (HAPI), and this is a mechanism being adopted at many data centers across Heliophysics and planetary science for the serving of time series data. Two existing tools are also being enhanced to read from HAPI servers, namely Autoplot from the University of Iowa and MIDL (Mission Independent Data Layer) from The Johns Hopkins Applied Physics Lab. Thus both tools will be able to access data from RPWS, MAG, CAPS, and MIMI. In addition to being able to access data from each other's institutions, these tools will be able to read from all the new datasets expected to come online using the HAPI standard in the near future. The PDS also plans to use HAPI for all the holdings at the Planetary and Plasma Interactions (PPI) node. A basic presentation of the new HAPI data server mechanism is presented, as is an early demonstration of the modified tools.