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1.  Exploring single-sample SNP and INDEL calling with whole-genome de novo assembly 
Bioinformatics  2012;28(14):1838-1844.
Motivation: Eugene Myers in his string graph paper suggested that in a string graph or equivalently a unitig graph, any path spells a valid assembly. As a string/unitig graph also encodes every valid assembly of reads, such a graph, provided that it can be constructed correctly, is in fact a lossless representation of reads. In principle, every analysis based on whole-genome shotgun sequencing (WGS) data, such as SNP and insertion/deletion (INDEL) calling, can also be achieved with unitigs.
Results: To explore the feasibility of using de novo assembly in the context of resequencing, we developed a de novo assembler, fermi, that assembles Illumina short reads into unitigs while preserving most of information of the input reads. SNPs and INDELs can be called by mapping the unitigs against a reference genome. By applying the method on 35-fold human resequencing data, we showed that in comparison to the standard pipeline, our approach yields similar accuracy for SNP calling and better results for INDEL calling. It has higher sensitivity than other de novo assembly based methods for variant calling. Our work suggests that variant calling with de novo assembly can be a beneficial complement to the standard variant calling pipeline for whole-genome resequencing. In the methodological aspects, we propose FMD-index for forward–backward extension of DNA sequences, a fast algorithm for finding all super-maximal exact matches and one-pass construction of unitigs from an FMD-index.
Availability: http://github.com/lh3/fermi
Contact: hengli@broadinstitute.org
doi:10.1093/bioinformatics/bts280
PMCID: PMC3389770  PMID: 22569178
2.  A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data 
Bioinformatics  2011;27(21):2987-2993.
Motivation: Most existing methods for DNA sequence analysis rely on accurate sequences or genotypes. However, in applications of the next-generation sequencing (NGS), accurate genotypes may not be easily obtained (e.g. multi-sample low-coverage sequencing or somatic mutation discovery). These applications press for the development of new methods for analyzing sequence data with uncertainty.
Results: We present a statistical framework for calling SNPs, discovering somatic mutations, inferring population genetical parameters and performing association tests directly based on sequencing data without explicit genotyping or linkage-based imputation. On real data, we demonstrate that our method achieves comparable accuracy to alternative methods for estimating site allele count, for inferring allele frequency spectrum and for association mapping. We also highlight the necessity of using symmetric datasets for finding somatic mutations and confirm that for discovering rare events, mismapping is frequently the leading source of errors.
Availability: http://samtools.sourceforge.net
Contact: hengli@broadinstitute.org
doi:10.1093/bioinformatics/btr509
PMCID: PMC3198575  PMID: 21903627
3.  Improving SNP discovery by base alignment quality 
Bioinformatics  2011;27(8):1157-1158.
Summary: I propose a new application of profile Hidden Markov Models in the area of SNP discovery from resequencing data, to greatly reduce false SNP calls caused by misalignments around insertions and deletions (indels). The central concept is per-Base Alignment Quality, which accurately measures the probability of a read base being wrongly aligned. The effectiveness of BAQ has been positively confirmed on large datasets by the 1000 Genomes Project analysis subgroup.
Availability: http://samtools.sourceforge.net
Contact: hengli@broadinstitute.org
doi:10.1093/bioinformatics/btr076
PMCID: PMC3072548  PMID: 21320865
4.  Tabix: fast retrieval of sequence features from generic TAB-delimited files 
Bioinformatics  2011;27(5):718-719.
Summary: Tabix is the first generic tool that indexes position sorted files in TAB-delimited formats such as GFF, BED, PSL, SAM and SQL export, and quickly retrieves features overlapping specified regions. Tabix features include few seek function calls per query, data compression with gzip compatibility and direct FTP/HTTP access. Tabix is implemented as a free command-line tool as well as a library in C, Java, Perl and Python. It is particularly useful for manually examining local genomic features on the command line and enables genome viewers to support huge data files and remote custom tracks over networks.
Availability and Implementation: http://samtools.sourceforge.net.
Contact: hengli@broadinstitute.org
doi:10.1093/bioinformatics/btq671
PMCID: PMC3042176  PMID: 21208982
5.  Fast and accurate long-read alignment with Burrows–Wheeler transform 
Bioinformatics  2010;26(5):589-595.
Motivation: Many programs for aligning short sequencing reads to a reference genome have been developed in the last 2 years. Most of them are very efficient for short reads but inefficient or not applicable for reads >200 bp because the algorithms are heavily and specifically tuned for short queries with low sequencing error rate. However, some sequencing platforms already produce longer reads and others are expected to become available soon. For longer reads, hashing-based software such as BLAT and SSAHA2 remain the only choices. Nonetheless, these methods are substantially slower than short-read aligners in terms of aligned bases per unit time.
Results: We designed and implemented a new algorithm, Burrows-Wheeler Aligner's Smith-Waterman Alignment (BWA-SW), to align long sequences up to 1 Mb against a large sequence database (e.g. the human genome) with a few gigabytes of memory. The algorithm is as accurate as SSAHA2, more accurate than BLAT, and is several to tens of times faster than both.
Availability: http://bio-bwa.sourceforge.net
Contact: rd@sanger.ac.uk
doi:10.1093/bioinformatics/btp698
PMCID: PMC2828108  PMID: 20080505
6.  The Sequence Alignment/Map format and SAMtools 
Bioinformatics  2009;25(16):2078-2079.
Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Availability: http://samtools.sourceforge.net
Contact: rd@sanger.ac.uk
doi:10.1093/bioinformatics/btp352
PMCID: PMC2723002  PMID: 19505943
7.  Detecting SNPs and estimating allele frequencies in clonal bacterial populations by sequencing pooled DNA 
Bioinformatics  2009;25(16):2074-2075.
Summary: 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 ≥80% sensitivity of SNP detection and strong correlation with true SNP frequency at poolwide read depth of 40×, declining only slightly at read depths 20–40×.
Availability: 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/.
Contact: kh2@sanger.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btp344
PMCID: PMC2722999  PMID: 19497932
8.  Fast and accurate short read alignment with Burrows–Wheeler transform 
Bioinformatics  2009;25(14):1754-1760.
Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals.
Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package.
Availability: http://maq.sourceforge.net
Contact: rd@sanger.ac.uk
doi:10.1093/bioinformatics/btp324
PMCID: PMC2705234  PMID: 19451168

Results 1-8 (8)