Base-calling of automated sequencer traces using Phred. I. accuracy assessment
TL;DR: In this article, a base-calling program for automated sequencer traces, phred, with improved accuracy was proposed. But it was not shown to achieve a lower error rate than the ABI software, averaging 40%-50% fewer errors in the data sets examined independent of position in read, machine running conditions, or sequencing chemistry.
Abstract: The availability of massive amounts of DNA sequence information has begun to revolutionize the practice of biology. As a result, current large-scale sequencing output, while impressive, is not adequate to keep pace with growing demand and, in particular, is far short of what will be required to obtain the 3-billion-base human genome sequence by the target date of 2005. To reach this goal, improved automation will be essential, and it is particularly important that human involvement in sequence data processing be significantly reduced or eliminated. Progress in this respect will require both improved accuracy of the data processing software and reliable accuracy measures to reduce the need for human involvement in error correction and make human review more efficient. Here, we describe one step toward that goal: a base-calling program for automated sequencer traces, phred, with improved accuracy. phred appears to be the first base-calling program to achieve a lower error rate than the ABI software, averaging 40%-50% fewer errors in the data sets examined independent of position in read, machine running conditions, or sequencing chemistry.
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TL;DR: The results of an international collaboration to produce and make freely available a draft sequence of the human genome are reported and an initial analysis is presented, describing some of the insights that can be gleaned from the sequence.
Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
22,269 citations
TL;DR: Bowtie extends previous Burrows-Wheeler techniques with a novel quality-aware backtracking algorithm that permits mismatches and can be used simultaneously to achieve even greater alignment speeds.
Abstract: Bowtie is an ultrafast, memory-efficient alignment program for aligning short DNA sequence reads to large genomes. For the human genome, Burrows-Wheeler indexing allows Bowtie to align more than 25 million reads per CPU hour with a memory footprint of approximately 1.3 gigabytes. Bowtie extends previous Burrows-Wheeler techniques with a novel quality-aware backtracking algorithm that permits mismatches. Multiple processor cores can be used simultaneously to achieve even greater alignment speeds. Bowtie is open source http://bowtie.cbcb.umd.edu.
20,335 citations
TL;DR: SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies.
Abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V−SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online (http://bioinf.spbau.ru/spades). It is distributed as open source software.
16,859 citations
Cites background from "Base-calling of automated sequencer..."
...(4) Stage 4 (contig construction) was well studied in the context of Sanger sequencing (Ewing et al., 1998)....
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...(4) Stage 4 (contig construction) was well studied in the context of Sanger sequencing (Ewing et al., 1998)....
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TL;DR: Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems are indicated.
Abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
12,098 citations
01 Jun 2012
TL;DR: SPAdes as mentioned in this paper is a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler and on popular assemblers Velvet and SoapDeNovo (for multicell data).
Abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.
10,124 citations
References
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TL;DR: A new method for determining nucleotide sequences in DNA is described, which makes use of the 2',3'-dideoxy and arabinon nucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.
Abstract: A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.
62,728 citations
"Base-calling of automated sequencer..." refers background or methods in this paper
...In better resolved regions of the trace, the most commonly seen electrophoretic anomalies are compressions (Sanger and Coulson 1975; Sanger et al. 1977), which occur when bases near the end of a single-stranded fragment bind to a complementary upstream region, creating a hairpin-like structure that…...
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...At present, nearly all DNA sequencing is done using the enzymatic dideoxy chain-termination method of Sanger (Sanger et al. 1977)....
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...I. Accuracy Assessment Brent Ewing,1 LaDeana Hillier,2 Michael C. Wendl,2 and Phil Green1,3 1Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195-7730 USA; 2Genome Sequencing Center, Washington University School of Medicine, Saint Louis, Missouri 63108 USA The…...
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...In better resolved regions of the trace, the most commonly seen electrophoretic anomalies are compressions (Sanger and Coulson 1975; Sanger et al. 1977), which occur when bases near the end of a single-stranded fragment bind to a complementary upstream region, creating a hairpin-like structure that migrates through the gel more rapidly than expected from its length, thus causing a peak to be shifted left of its expected position....
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...I. Accuracy Assessment Brent Ewing,1 LaDeana Hillier,2 Michael C. Wendl,2 and Phil Green1,3 1Department of Molecular Biotechnology, University of Washington, Seattle, Washington 98195-7730 USA; 2Genome Sequencing Center, Washington University School of Medicine, Saint Louis, Missouri 63108 USA The availability of massive amounts of DNA sequence information has begun to revolutionize the practice of biology....
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01 Jan 1994
TL;DR: The Diskette v 2.06, 3.5''[1.44M] for IBM PC, PS/2 and compatibles [DOS] Reference Record created on 2004-09-07, modified on 2016-08-08.
Abstract: Note: Includes bibliographical references, 3 appendixes and 2 indexes.- Diskette v 2.06, 3.5''[1.44M] for IBM PC, PS/2 and compatibles [DOS] Reference Record created on 2004-09-07, modified on 2016-08-08
19,881 citations
Book•
31 Jan 1986TL;DR: Numerical Recipes: The Art of Scientific Computing as discussed by the authors is a complete text and reference book on scientific computing with over 100 new routines (now well over 300 in all), plus upgraded versions of many of the original routines, with many new topics presented at the same accessible level.
Abstract: From the Publisher:
This is the revised and greatly expanded Second Edition of the hugely popular Numerical Recipes: The Art of Scientific Computing. The product of a unique collaboration among four leading scientists in academic research and industry, Numerical Recipes is a complete text and reference book on scientific computing. In a self-contained manner it proceeds from mathematical and theoretical considerations to actual practical computer routines. With over 100 new routines (now well over 300 in all), plus upgraded versions of many of the original routines, this book is more than ever the most practical, comprehensive handbook of scientific computing available today. The book retains the informal, easy-to-read style that made the first edition so popular, with many new topics presented at the same accessible level. In addition, some sections of more advanced material have been introduced, set off in small type from the main body of the text. Numerical Recipes is an ideal textbook for scientists and engineers and an indispensable reference for anyone who works in scientific computing. Highlights of the new material include a new chapter on integral equations and inverse methods; multigrid methods for solving partial differential equations; improved random number routines; wavelet transforms; the statistical bootstrap method; a new chapter on "less-numerical" algorithms including compression coding and arbitrary precision arithmetic; band diagonal linear systems; linear algebra on sparse matrices; Cholesky and QR decomposition; calculation of numerical derivatives; Pade approximants, and rational Chebyshev approximation; new special functions; Monte Carlo integration in high-dimensional spaces; globally convergent methods for sets of nonlinear equations; an expanded chapter on fast Fourier methods; spectral analysis on unevenly sampled data; Savitzky-Golay smoothing filters; and two-dimensional Kolmogorov-Smirnoff tests. All this is in addition to material on such basic top
12,662 citations
11,285 citations
"Base-calling of automated sequencer..." refers methods in this paper
...Then, among all sine waves having a period in the permitted set, the one having the GENOME RESEARCH 177 largest inner product with the damped trace is found, using simple Fourier methods (Press et al. 1988)....
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TL;DR: This letter extends the heuristic homology algorithm of Needleman & Wunsch (1970) to find a pair of segments, one from each of two long sequences, such that there is no other Pair of segments with greater similarity (homology).
10,262 citations
"Base-calling of automated sequencer..." refers methods in this paper
...Alignment of the reads to the finished sequence was performed by use of a restricted Smith– Waterman (Smith and Waterman 1981) algorithm as implemented in the program cross match (P. Green, in prep.)...
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...Alignment of the reads to the finished sequence was performed by use of a restricted Smith‐ Waterman ( Smith and Waterman 1981 ) algorithm as implemented in the program cross match (P. Green, in prep.), which searches bands in the Smith- ‐Waterman matrix surrounding word matches between the query and target sequences....
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