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1.  ARYANA: Aligning Reads by Yet Another Approach 
BMC Bioinformatics  2014;15(Suppl 9):S12.
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
doi:10.1186/1471-2105-15-S9-S12
PMCID: PMC4168712  PMID: 25252881
Alignment; Read mapping; DNA sequencing; Next generation sequencing (NGS)
2.  CloudAligner: A fast and full-featured MapReduce based tool for sequence mapping 
BMC Research Notes  2011;4:171.
Background
Research in genetics has developed rapidly recently due to the aid of next generation sequencing (NGS). However, massively-parallel NGS produces enormous amounts of data, which leads to storage, compatibility, scalability, and performance issues. The Cloud Computing and MapReduce framework, which utilizes hundreds or thousands of shared computers to map sequencing reads quickly and efficiently to reference genome sequences, appears to be a very promising solution for these issues. Consequently, it has been adopted by many organizations recently, and the initial results are very promising. However, since these are only initial steps toward this trend, the developed software does not provide adequate primary functions like bisulfite, pair-end mapping, etc., in on-site software such as RMAP or BS Seeker. In addition, existing MapReduce-based applications were not designed to process the long reads produced by the most recent second-generation and third-generation NGS instruments and, therefore, are inefficient. Last, it is difficult for a majority of biologists untrained in programming skills to use these tools because most were developed on Linux with a command line interface.
Results
To urge the trend of using Cloud technologies in genomics and prepare for advances in second- and third-generation DNA sequencing, we have built a Hadoop MapReduce-based application, CloudAligner, which achieves higher performance, covers most primary features, is more accurate, and has a user-friendly interface. It was also designed to be able to deal with long sequences. The performance gain of CloudAligner over Cloud-based counterparts (35 to 80%) mainly comes from the omission of the reduce phase. In comparison to local-based approaches, the performance gain of CloudAligner is from the partition and parallel processing of the huge reference genome as well as the reads. The source code of CloudAligner is available at http://cloudaligner.sourceforge.net/ and its web version is at http://mine.cs.wayne.edu:8080/CloudAligner/.
Conclusions
Our results show that CloudAligner is faster than CloudBurst, provides more accurate results than RMAP, and supports various input as well as output formats. In addition, with the web-based interface, it is easier to use than its counterparts.
doi:10.1186/1756-0500-4-171
PMCID: PMC3127959  PMID: 21645377
3.  How do alignment programs perform on sequencing data with varying qualities and from repetitive regions? 
BioData Mining  2012;5:6.
Background
Next-generation sequencing technologies generate a significant number of short reads that are utilized to address a variety of biological questions. However, quite often, sequencing reads tend to have low quality at the 3’ end and are generated from the repetitive regions of a genome. It is unclear how different alignment programs perform under these different cases. In order to investigate this question, we use both real data and simulated data with the above issues to evaluate the performance of four commonly used algorithms: SOAP2, Bowtie, BWA, and Novoalign.
Methods
The performance of different alignment algorithms are measured in terms of concordance between any pair of aligners (for real sequencing data without known truth) and the accuracy of simulated read alignment.
Results
Our results show that, for sequencing data with reads that have relatively good quality or that have had low quality bases trimmed off, all four alignment programs perform similarly. We have also demonstrated that trimming off low quality ends markedly increases the number of aligned reads and improves the consistency among different aligners as well, especially for low quality data. However, Novoalign is more sensitive to the improvement of data quality. Trimming off low quality ends significantly increases the concordance between Novoalign and other aligners. As for aligning reads from repetitive regions, our simulation data show that reads from repetitive regions tend to be aligned incorrectly, and suppressing reads with multiple hits can improve alignment accuracy.
Conclusions
This study provides a systematic comparison of commonly used alignment algorithms in the context of sequencing data with varying qualities and from repetitive regions. Our approach can be applied to different sequencing data sets generated from different platforms. It can also be utilized to study the performance of other alignment programs.
doi:10.1186/1756-0381-5-6
PMCID: PMC3414812  PMID: 22709551
Next generation sequencing; Alignment; Sequencing quality; SOAP2; Bowtie; BWA; Novoalign
4.  GASSST: global alignment short sequence search tool 
Bioinformatics  2010;26(20):2534-2540.
Motivation: The rapid development of next-generation sequencing technologies able to produce huge amounts of sequence data is leading to a wide range of new applications. This triggers the need for fast and accurate alignment software. Common techniques often restrict indels in the alignment to improve speed, whereas more flexible aligners are too slow for large-scale applications. Moreover, many current aligners are becoming inefficient as generated reads grow ever larger. Our goal with our new aligner GASSST (Global Alignment Short Sequence Search Tool) is thus 2-fold—achieving high performance with no restrictions on the number of indels with a design that is still effective on long reads.
Results: We propose a new efficient filtering step that discards most alignments coming from the seed phase before they are checked by the costly dynamic programming algorithm. We use a carefully designed series of filters of increasing complexity and efficiency to quickly eliminate most candidate alignments in a wide range of configurations. The main filter uses a precomputed table containing the alignment score of short four base words aligned against each other. This table is reused several times by a new algorithm designed to approximate the score of the full dynamic programming algorithm. We compare the performance of GASSST against BWA, BFAST, SSAHA2 and PASS. We found that GASSST achieves high sensitivity in a wide range of configurations and faster overall execution time than other state-of-the-art aligners.
Availability: GASSST is distributed under the CeCILL software license at http://www.irisa.fr/symbiose/projects/gassst/
Contact: guillaume.rizk@irisa.fr; dominique.lavenier@irisa.fr
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq485
PMCID: PMC2951093  PMID: 20739310
5.  A de novo next generation genomic sequence assembler based on string graph and MapReduce cloud computing framework 
BMC Genomics  2012;13(Suppl 7):S28.
Background
State-of-the-art high-throughput sequencers, e.g., the Illumina HiSeq series, generate sequencing reads that are longer than 150 bp up to a total of 600 Gbp of data per run. The high-throughput sequencers generate lengthier reads with greater sequencing depth than those generated by previous technologies. Two major challenges exist in using the high-throughput technology for de novo assembly of genomes. First, the amount of physical memory may be insufficient to store the data structure of the assembly algorithm, even for high-end multicore processors. Moreover, the graph-theoretical model used to capture intersection relationships of the reads may contain structural defects that are not well managed by existing assembly algorithms.
Results
We developed a distributed genome assembler based on string graphs and MapReduce framework, known as the CloudBrush. The assembler includes a novel edge-adjustment algorithm to detect structural defects by examining the neighboring reads of a specific read for sequencing errors and adjusting the edges of the string graph, if necessary. CloudBrush is evaluated against GAGE benchmarks to compare its assembly quality with the other assemblers. The results show that our assemblies have a moderate N50, a low misassembly rate of misjoins, and indels of > 5 bp. In addition, we have introduced two measures, known as precision and recall, to address the issues of faithfully aligned contigs to target genomes. Compared with the assembly tools used in the GAGE benchmarks, CloudBrush is shown to produce contigs with high precision and recall. We also verified the effectiveness of the edge-adjustment algorithm using simulated datasets and ran CloudBrush on a nematode dataset using a commercial cloud. CloudBrush assembler is available at https://github.com/ice91/CloudBrush.
doi:10.1186/1471-2164-13-S7-S28
PMCID: PMC3521391  PMID: 23282094
6.  CloudBurst: highly sensitive read mapping with MapReduce 
Bioinformatics  2009;25(11):1363-1369.
Motivation: Next-generation DNA sequencing machines are generating an enormous amount of sequence data, placing unprecedented demands on traditional single-processor read-mapping algorithms. CloudBurst is a new parallel read-mapping algorithm optimized for mapping next-generation sequence data to the human genome and other reference genomes, for use in a variety of biological analyses including SNP discovery, genotyping and personal genomics. It is modeled after the short read-mapping program RMAP, and reports either all alignments or the unambiguous best alignment for each read with any number of mismatches or differences. This level of sensitivity could be prohibitively time consuming, but CloudBurst uses the open-source Hadoop implementation of MapReduce to parallelize execution using multiple compute nodes.
Results: CloudBurst's running time scales linearly with the number of reads mapped, and with near linear speedup as the number of processors increases. In a 24-processor core configuration, CloudBurst is up to 30 times faster than RMAP executing on a single core, while computing an identical set of alignments. Using a larger remote compute cloud with 96 cores, CloudBurst improved performance by >100-fold, reducing the running time from hours to mere minutes for typical jobs involving mapping of millions of short reads to the human genome.
Availability: CloudBurst is available open-source as a model for parallelizing algorithms with MapReduce at http://cloudburst-bio.sourceforge.net/.
Contact: mschatz@umiacs.umd.edu
doi:10.1093/bioinformatics/btp236
PMCID: PMC2682523  PMID: 19357099
7.  Evaluation of Alignment Algorithms for Discovery and Identification of Pathogens Using RNA-Seq 
PLoS ONE  2013;8(10):e76935.
Next-generation sequencing technologies provide an unparallelled opportunity for the characterization and discovery of known and novel viruses. Because viruses are known to have the highest mutation rates when compared to eukaryotic and bacterial organisms, we assess the extent to which eleven well-known alignment algorithms (BLAST, BLAT, BWA, BWA-SW, BWA-MEM, BFAST, Bowtie2, Novoalign, GSNAP, SHRiMP2 and STAR) can be used for characterizing mutated and non-mutated viral sequences - including those that exhibit RNA splicing - in transcriptome samples. To evaluate aligners objectively we developed a realistic RNA-Seq simulation and evaluation framework (RiSER) and propose a new combined score to rank aligners for viral characterization in terms of their precision, sensitivity and alignment accuracy. We used RiSER to simulate both human and viral read sequences and suggest the best set of aligners for viral sequence characterization in human transcriptome samples. Our results show that significant and substantial differences exist between aligners and that a digital-subtraction-based viral identification framework can and should use different aligners for different parts of the process. We determine the extent to which mutated viral sequences can be effectively characterized and show that more sensitive aligners such as BLAST, BFAST, SHRiMP2, BWA-SW and GSNAP can accurately characterize substantially divergent viral sequences with up to 15% overall sequence mutation rate. We believe that the results presented here will be useful to researchers choosing aligners for viral sequence characterization using next-generation sequencing data.
doi:10.1371/journal.pone.0076935
PMCID: PMC3813700  PMID: 24204709
8.  Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM) 
Bioinformatics  2011;27(18):2518-2528.
Motivation: A critical task in high-throughput sequencing is aligning millions of short reads to a reference genome. Alignment is especially complicated for RNA sequencing (RNA-Seq) because of RNA splicing. A number of RNA-Seq algorithms are available, and claim to align reads with high accuracy and efficiency while detecting splice junctions. RNA-Seq data are discrete in nature; therefore, with reasonable gene models and comparative metrics RNA-Seq data can be simulated to sufficient accuracy to enable meaningful benchmarking of alignment algorithms. The exercise to rigorously compare all viable published RNA-Seq algorithms has not been performed previously.
Results: We developed an RNA-Seq simulator that models the main impediments to RNA alignment, including alternative splicing, insertions, deletions, substitutions, sequencing errors and intron signal. We used this simulator to measure the accuracy and robustness of available algorithms at the base and junction levels. Additionally, we used reverse transcription–polymerase chain reaction (RT–PCR) and Sanger sequencing to validate the ability of the algorithms to detect novel transcript features such as novel exons and alternative splicing in RNA-Seq data from mouse retina. A pipeline based on BLAT was developed to explore the performance of established tools for this problem, and to compare it to the recently developed methods. This pipeline, the RNA-Seq Unified Mapper (RUM), performs comparably to the best current aligners and provides an advantageous combination of accuracy, speed and usability.
Availability: The RUM pipeline is distributed via the Amazon Cloud and for computing clusters using the Sun Grid Engine (http://cbil.upenn.edu/RUM).
Contact: ggrant@pcbi.upenn.edu; epierce@mail.med.upenn.edu
Supplementary Information:The RNA-Seq sequence reads described in the article are deposited at GEO, accession GSE26248.
doi:10.1093/bioinformatics/btr427
PMCID: PMC3167048  PMID: 21775302
9.  AlignGraph: algorithm for secondary de novo genome assembly guided by closely related references 
Bioinformatics  2014;30(12):i319-i328.
Motivation: De novo assemblies of genomes remain one of the most challenging applications in next-generation sequencing. Usually, their results are incomplete and fragmented into hundreds of contigs. Repeats in genomes and sequencing errors are the main reasons for these complications. With the rapidly growing number of sequenced genomes, it is now feasible to improve assemblies by guiding them with genomes from related species.
Results: Here we introduce AlignGraph, an algorithm for extending and joining de novo-assembled contigs or scaffolds guided by closely related reference genomes. It aligns paired-end (PE) reads and preassembled contigs or scaffolds to a close reference. From the obtained alignments, it builds a novel data structure, called the PE multipositional de Bruijn graph. The incorporated positional information from the alignments and PE reads allows us to extend the initial assemblies, while avoiding incorrect extensions and early terminations. In our performance tests, AlignGraph was able to substantially improve the contigs and scaffolds from several assemblers. For instance, 28.7–62.3% of the contigs of Arabidopsis thaliana and human could be extended, resulting in improvements of common assembly metrics, such as an increase of the N50 of the extendable contigs by 89.9–94.5% and 80.3–165.8%, respectively. In another test, AlignGraph was able to improve the assembly of a published genome (Arabidopsis strain Landsberg) by increasing the N50 of its extendable scaffolds by 86.6%. These results demonstrate AlignGraph’s efficiency in improving genome assemblies by taking advantage of closely related references.
Availability and implementation: The AlignGraph software can be downloaded for free from this site: https://github.com/baoe/AlignGraph.
Contact: thomas.girke@ucr.edu
doi:10.1093/bioinformatics/btu291
PMCID: PMC4058956  PMID: 24932000
10.  Evaluation and Comparison of Multiple Aligners for Next-Generation Sequencing Data Analysis 
BioMed Research International  2014;2014:309650.
Next-generation sequencing (NGS) technology has rapidly advanced and generated the massive data volumes. To align and map the NGS data, biologists often randomly select a number of aligners without concerning their suitable feature, high performance, and high accuracy as well as sequence variations and polymorphisms existing on reference genome. This study aims to systematically evaluate and compare the capability of multiple aligners for NGS data analysis. To explore this capability, we firstly performed alignment algorithms comparison and classification. We further used long-read and short-read datasets from both real-life and in silico NGS data for comparative analysis and evaluation of these aligners focusing on three criteria, namely, application-specific alignment feature, computational performance, and alignment accuracy. Our study demonstrated the overall evaluation and comparison of multiple aligners for NGS data analysis. This serves as an important guiding resource for biologists to gain further insight into suitable selection of aligners for specific and broad applications.
doi:10.1155/2014/309650
PMCID: PMC3980841  PMID: 24779008
11.  Discovering Transcription Factor Binding Sites in Highly Repetitive Regions of Genomes with Multi-Read Analysis of ChIP-Seq Data 
PLoS Computational Biology  2011;7(7):e1002111.
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.
Author Summary
Annotating repetitive regions of genomes experimentally is a challenging task. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) provides valuable data for characterizing repetitive regions of genomes in terms of transcription factor binding. Although ChIP-seq technology has been maturing, available ChIP-seq analysis methods and software rely on discarding sequence reads that map to multiple locations on the reference genome (multi-reads), thereby generating a missed opportunity for assessing transcription factor binding to highly repetitive regions of genomes. We develop a computational algorithm that takes multi-reads into account in ChIP-seq analysis. We show with computational experiments that multi-reads lead to significant increase in sequencing depths and identification of binding regions that are otherwise not identifiable when only reads that uniquely map to the reference genome (uni-reads) are used. In particular, we show that the number of binding regions identified can increase up to 36%. We support our computational predictions with independent quantitative real-time ChIP validation of binding regions identified only when multi-reads are incorporated in the analysis of a mouse GATA1 ChIP-seq experiment.
doi:10.1371/journal.pcbi.1002111
PMCID: PMC3136429  PMID: 21779159
12.  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
13.  Separating metagenomic short reads into genomes via clustering 
Background
The metagenomics approach allows the simultaneous sequencing of all genomes in an environmental sample. This results in high complexity datasets, where in addition to repeats and sequencing errors, the number of genomes and their abundance ratios are unknown. Recently developed next-generation sequencing (NGS) technologies significantly improve the sequencing efficiency and cost. On the other hand, they result in shorter reads, which makes the separation of reads from different species harder. Among the existing computational tools for metagenomic analysis, there are similarity-based methods that use reference databases to align reads and composition-based methods that use composition patterns (i.e., frequencies of short words or l-mers) to cluster reads. Similarity-based methods are unable to classify reads from unknown species without close references (which constitute the majority of reads). Since composition patterns are preserved only in significantly large fragments, composition-based tools cannot be used for very short reads, which becomes a significant limitation with the development of NGS. A recently proposed algorithm, AbundanceBin, introduced another method that bins reads based on predicted abundances of the genomes sequenced. However, it does not separate reads from genomes of similar abundance levels.
Results
In this work, we present a two-phase heuristic algorithm for separating short paired-end reads from different genomes in a metagenomic dataset. We use the observation that most of the l-mers belong to unique genomes when l is sufficiently large. The first phase of the algorithm results in clusters of l-mers each of which belongs to one genome. During the second phase, clusters are merged based on l-mer repeat information. These final clusters are used to assign reads. The algorithm could handle very short reads and sequencing errors. It is initially designed for genomes with similar abundance levels and then extended to handle arbitrary abundance ratios. The software can be download for free at http://www.cs.ucr.edu/∼tanaseio/toss.htm.
Conclusions
Our tests on a large number of simulated metagenomic datasets concerning species at various phylogenetic distances demonstrate that genomes can be separated if the number of common repeats is smaller than the number of genome-specific repeats. For such genomes, our method can separate NGS reads with a high precision and sensitivity.
doi:10.1186/1748-7188-7-27
PMCID: PMC3537596  PMID: 23009059
Metagenomics; NGS short reads; Genome separation; Clustering
14.  Fast Mapping of Short Sequences with Mismatches, Insertions and Deletions Using Index Structures 
PLoS Computational Biology  2009;5(9):e1000502.
With few exceptions, current methods for short read mapping make use of simple seed heuristics to speed up the search. Most of the underlying matching models neglect the necessity to allow not only mismatches, but also insertions and deletions. Current evaluations indicate, however, that very different error models apply to the novel high-throughput sequencing methods. While the most frequent error-type in Illumina reads are mismatches, reads produced by 454's GS FLX predominantly contain insertions and deletions (indels). Even though 454 sequencers are able to produce longer reads, the method is frequently applied to small RNA (miRNA and siRNA) sequencing. Fast and accurate matching in particular of short reads with diverse errors is therefore a pressing practical problem. We introduce a matching model for short reads that can, besides mismatches, also cope with indels. It addresses different error models. For example, it can handle the problem of leading and trailing contaminations caused by primers and poly-A tails in transcriptomics or the length-dependent increase of error rates. In these contexts, it thus simplifies the tedious and error-prone trimming step. For efficient searches, our method utilizes index structures in the form of enhanced suffix arrays. In a comparison with current methods for short read mapping, the presented approach shows significantly increased performance not only for 454 reads, but also for Illumina reads. Our approach is implemented in the software segemehl available at http://www.bioinf.uni-leipzig.de/Software/segemehl/.
Author Summary
The successful mapping of high-throughput sequencing (HTS) reads to reference genomes largely depends on the accuracy of both the sequencing technologies and reference genomes. Current mapping algorithms focus on mapping with mismatches but largely neglect insertions and deletions—regardless of whether they are caused by sequencing errors or genomic variation. Furthermore, trailing contaminations by primers and declining read qualities can be cumbersome for programs that allow a maximum number of mismatches. We have developed and implemented a new approach for short read mapping that, in a first step, computes exact matches of the read and the reference genome. The exact matches are then modified by a limited number of mismatches, insertions and deletions. From the set of exact and inexact matches, we select those with minimum score-based E-values. This gives a set of regions in the reference genome which is aligned to the read using Myers bitvector algorithm [1]. Our method utilizes enhanced suffix arrays [2] to quickly find the exact and inexact matches. It maps more reads and achieves higher recall rates than previous methods. This consistently holds for reads produced by 454 as well as Illumina sequencing technologies.
doi:10.1371/journal.pcbi.1000502
PMCID: PMC2730575  PMID: 19750212
15.  BamView: visualizing and interpretation of next-generation sequencing read alignments 
Briefings in Bioinformatics  2012;14(2):203-212.
So-called next-generation sequencing (NGS) has provided the ability to sequence on a massive scale at low cost, enabling biologists to perform powerful experiments and gain insight into biological processes. BamView has been developed to visualize and analyse sequence reads from NGS platforms, which have been aligned to a reference sequence. It is a desktop application for browsing the aligned or mapped reads [Ruffalo, M, LaFramboise, T, Koyutürk, M. Comparative analysis of algorithms for next-generation sequencing read alignment. Bioinformatics 2011;27:2790–6] at different levels of magnification, from nucleotide level, where the base qualities can be seen, to genome or chromosome level where overall coverage is shown. To enable in-depth investigation of NGS data, various views are provided that can be configured to highlight interesting aspects of the data. Multiple read alignment files can be overlaid to compare results from different experiments, and filters can be applied to facilitate the interpretation of the aligned reads. As well as being a standalone application it can be used as an integrated part of the Artemis genome browser, BamView allows the user to study NGS data in the context of the sequence and annotation of the reference genome. Single nucleotide polymorphism (SNP) density and candidate SNP sites can be highlighted and investigated, and read-pair information can be used to discover large structural insertions and deletions. The application will also calculate simple analyses of the read mapping, including reporting the read counts and reads per kilobase per million mapped reads (RPKM) for genes selected by the user.
Availability: BamView and Artemis are freely available software. These can be downloaded from their home pages:
http://bamview.sourceforge.net/; http://www.sanger.ac.uk/resources/software/artemis/.
Requirements: Java 1.6 or higher.
doi:10.1093/bib/bbr073
PMCID: PMC3603209  PMID: 22253280
genome browser; next-generation sequencing; visualization; Artemis; BamView
16.  SHRiMP: Accurate Mapping of Short Color-space Reads 
PLoS Computational Biology  2009;5(5):e1000386.
The development of Next Generation Sequencing technologies, capable of sequencing hundreds of millions of short reads (25–70 bp each) in a single run, is opening the door to population genomic studies of non-model species. In this paper we present SHRiMP - the SHort Read Mapping Package: a set of algorithms and methods to map short reads to a genome, even in the presence of a large amount of polymorphism. Our method is based upon a fast read mapping technique, separate thorough alignment methods for regular letter-space as well as AB SOLiD (color-space) reads, and a statistical model for false positive hits. We use SHRiMP to map reads from a newly sequenced Ciona savignyi individual to the reference genome. We demonstrate that SHRiMP can accurately map reads to this highly polymorphic genome, while confirming high heterozygosity of C. savignyi in this second individual. SHRiMP is freely available at http://compbio.cs.toronto.edu/shrimp.
Author Summary
Next Generation Sequencing (NGS) technologies are revolutionizing the way biologists acquire and analyze genomic data. NGS machines, such as Illumina/Solexa and AB SOLiD, are able to sequence genomes more cheaply by 200-fold than previous methods. One of the main application areas of NGS technologies is the discovery of genomic variation within a given species. The first step in discovering this variation is the mapping of reads sequenced from a donor individual to a known (“reference”) genome. Differences between the reference and the reads are indicative either of polymorphisms, or of sequencing errors. Since the introduction of NGS technologies, many methods have been devised for mapping reads to reference genomes. However, these algorithms often sacrifice sensitivity for fast running time. While they are successful at mapping reads from organisms that exhibit low polymorphism rates, they do not perform well at mapping reads from highly polymorphic organisms. We present a novel read mapping method, SHRiMP, that can handle much greater amounts of polymorphism. Using Ciona savignyi as our target organism, we demonstrate that our method discovers significantly more variation than other methods. Additionally, we develop color-space extensions to classical alignment algorithms, allowing us to map color-space, or “dibase”, reads generated by AB SOLiD sequencers.
doi:10.1371/journal.pcbi.1000386
PMCID: PMC2678294  PMID: 19461883
17.  Oculus: faster sequence alignment by streaming read compression 
BMC Bioinformatics  2012;13:297.
Background
Despite significant advancement in alignment algorithms, the exponential growth of nucleotide sequencing throughput threatens to outpace bioinformatic analysis. Computation may become the bottleneck of genome analysis if growing alignment costs are not mitigated by further improvement in algorithms. Much gain has been gleaned from indexing and compressing alignment databases, but many widely used alignment tools process input reads sequentially and are oblivious to any underlying redundancy in the reads themselves.
Results
Here we present Oculus, a software package that attaches to standard aligners and exploits read redundancy by performing streaming compression, alignment, and decompression of input sequences. This nearly lossless process (> 99.9%) led to alignment speedups of up to 270% across a variety of data sets, while requiring a modest amount of memory. We expect that streaming read compressors such as Oculus could become a standard addition to existing RNA-Seq and ChIP-Seq alignment pipelines, and potentially other applications in the future as throughput increases.
Conclusions
Oculus efficiently condenses redundant input reads and wraps existing aligners to provide nearly identical SAM output in a fraction of the aligner runtime. It includes a number of useful features, such as tunable performance and fidelity options, compatibility with FASTA or FASTQ files, and adherence to the SAM format. The platform-independent C++ source code is freely available online, at http://code.google.com/p/oculus-bio.
doi:10.1186/1471-2105-13-297
PMCID: PMC3534618  PMID: 23148484
DNA nucleotide sequence alignment streaming identity redundancy compression software algorithm
18.  Coval: Improving Alignment Quality and Variant Calling Accuracy for Next-Generation Sequencing Data 
PLoS ONE  2013;8(10):e75402.
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/.
doi:10.1371/journal.pone.0075402
PMCID: PMC3792961  PMID: 24116042
19.  Fast Statistical Alignment 
PLoS Computational Biology  2009;5(5):e1000392.
We describe a new program for the alignment of multiple biological sequences that is both statistically motivated and fast enough for problem sizes that arise in practice. Our Fast Statistical Alignment program is based on pair hidden Markov models which approximate an insertion/deletion process on a tree and uses a sequence annealing algorithm to combine the posterior probabilities estimated from these models into a multiple alignment. FSA uses its explicit statistical model to produce multiple alignments which are accompanied by estimates of the alignment accuracy and uncertainty for every column and character of the alignment—previously available only with alignment programs which use computationally-expensive Markov Chain Monte Carlo approaches—yet can align thousands of long sequences. Moreover, FSA utilizes an unsupervised query-specific learning procedure for parameter estimation which leads to improved accuracy on benchmark reference alignments in comparison to existing programs. The centroid alignment approach taken by FSA, in combination with its learning procedure, drastically reduces the amount of false-positive alignment on biological data in comparison to that given by other methods. The FSA program and a companion visualization tool for exploring uncertainty in alignments can be used via a web interface at http://orangutan.math.berkeley.edu/fsa/, and the source code is available at http://fsa.sourceforge.net/.
Author Summary
Biological sequence alignment is one of the fundamental problems in comparative genomics, yet it remains unsolved. Over sixty sequence alignment programs are listed on Wikipedia, and many new programs are published every year. However, many popular programs suffer from pathologies such as aligning unrelated sequences and producing discordant alignments in protein (amino acid) and codon (nucleotide) space, casting doubt on the accuracy of the inferred alignments. Inaccurate alignments can introduce large and unknown systematic biases into downstream analyses such as phylogenetic tree reconstruction and substitution rate estimation. We describe a new program for multiple sequence alignment which can align protein, RNA and DNA sequence and improves on the accuracy of existing approaches on benchmarks of protein and RNA structural alignments and simulated mammalian and fly genomic alignments. Our approach, which seeks to find the alignment which is closest to the truth under our statistical model, leaves unrelated sequences largely unaligned and produces concordant alignments in protein and codon space. It is fast enough for difficult problems such as aligning orthologous genomic regions or aligning hundreds or thousands of proteins. It furthermore has a companion GUI for visualizing the estimated alignment reliability.
doi:10.1371/journal.pcbi.1000392
PMCID: PMC2684580  PMID: 19478997
20.  STAR: ultrafast universal RNA-seq aligner 
Bioinformatics  2012;29(1):15-21.
Motivation: Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases.
Results: To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80–90% success rate, corroborating the high precision of the STAR mapping strategy.
Availability and implementation: STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.
Contact: dobin@cshl.edu.
doi:10.1093/bioinformatics/bts635
PMCID: PMC3530905  PMID: 23104886
21.  A Rank-Based Sequence Aligner with Applications in Phylogenetic Analysis 
PLoS ONE  2014;9(8):e104006.
Recent tools for aligning short DNA reads have been designed to optimize the trade-off between correctness and speed. This paper introduces a method for assigning a set of short DNA reads to a reference genome, under Local Rank Distance (LRD). The rank-based aligner proposed in this work aims to improve correctness over speed. However, some indexing strategies to speed up the aligner are also investigated. The LRD aligner is improved in terms of speed by storing -mer positions in a hash table for each read. Another improvement, that produces an approximate LRD aligner, is to consider only the positions in the reference that are likely to represent a good positional match of the read. The proposed aligner is evaluated and compared to other state of the art alignment tools in several experiments. A set of experiments are conducted to determine the precision and the recall of the proposed aligner, in the presence of contaminated reads. In another set of experiments, the proposed aligner is used to find the order, the family, or the species of a new (or unknown) organism, given only a set of short Next-Generation Sequencing DNA reads. The empirical results show that the aligner proposed in this work is highly accurate from a biological point of view. Compared to the other evaluated tools, the LRD aligner has the important advantage of being very accurate even for a very low base coverage. Thus, the LRD aligner can be considered as a good alternative to standard alignment tools, especially when the accuracy of the aligner is of high importance. Source code and UNIX binaries of the aligner are freely available for future development and use at http://lrd.herokuapp.com/aligners. The software is implemented in C++ and Java, being supported on UNIX and MS Windows.
doi:10.1371/journal.pone.0104006
PMCID: PMC4136772  PMID: 25133391
22.  CLAST: CUDA implemented large-scale alignment search tool 
BMC Bioinformatics  2014;15(1):406.
Background
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.
Results
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.
Conclusions
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0406-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12859-014-0406-y
PMCID: PMC4271471  PMID: 25495907
GPU; CUDA; Metagenomics; Microbial community; Sequence similarity search tool
23.  RandAL: a randomized approach to aligning DNA sequences to reference genomes 
BMC Genomics  2014;15(Suppl 5):S2.
Background
The alignment of short reads generated by next-generation sequencers to genomes is an important problem in many biomedical and bioinformatics applications. Although many proposed methods work very well on narrow ranges of read lengths, they tend to suffer in performance and alignment quality for reads outside of these ranges.
Results
We introduce RandAL, a novel method that aligns DNA sequences to reference genomes. Our approach utilizes two FM indices to facilitate efficient bidirectional searching, a pruning heuristic to speed up the computing of edit distances, and most importantly, a randomized strategy that enables effective estimation of key parameters. Extensive comparisons showed that RandAL outperformed popular aligners in most instances and was unique in its consistent and accurate performance over a wide range of read lengths and error rates. The software package is publicly available at https://github.com/namsyvo/RandAL.
Conclusions
RandAL promises to align effectively and accurately short reads that come from a variety of technologies with different read lengths and rates of sequencing error.
doi:10.1186/1471-2164-15-S5-S2
PMCID: PMC4120144  PMID: 25081493
next-gen sequencing; short read alignment; randomization
24.  A High-Throughput DNA Sequence Aligner for Microbial Ecology Studies 
PLoS ONE  2009;4(12):e8230.
As the scope of microbial surveys expands with the parallel growth in sequencing capacity, a significant bottleneck in data analysis is the ability to generate a biologically meaningful multiple sequence alignment. The most commonly used aligners have varying alignment quality and speed, tend to depend on a specific reference alignment, or lack a complete description of the underlying algorithm. The purpose of this study was to create and validate an aligner with the goal of quickly generating a high quality alignment and having the flexibility to use any reference alignment. Using the simple nearest alignment space termination algorithm, the resulting aligner operates in linear time, requires a small memory footprint, and generates a high quality alignment. In addition, the alignments generated for variable regions were of as high a quality as the alignment of full-length sequences. As implemented, the method was able to align 18 full-length 16S rRNA gene sequences and 58 V2 region sequences per second to the 50,000-column SILVA reference alignment. Most importantly, the resulting alignments were of a quality equal to SILVA-generated alignments. The aligner described in this study will enable scientists to rapidly generate robust multiple sequences alignments that are implicitly based upon the predicted secondary structure of the 16S rRNA molecule. Furthermore, because the implementation is not connected to a specific database it is easy to generalize the method to reference alignments for any DNA sequence.
doi:10.1371/journal.pone.0008230
PMCID: PMC2788221  PMID: 20011594
25.  Assessment of the Impact of Using a Reference Transcriptome in Mapping Short RNA-Seq Reads 
PLoS ONE  2014;9(7):e101374.
RNA-Seq has become increasingly popular in transcriptome profiling. The major challenge in RNA-Seq data analysis is the accurate mapping of junction reads to their genomic origins. To detect splicing sites in short reads, many RNA-Seq aligners use reference transcriptome to inform placement of junction reads. However, no systematic evaluation has been performed to assess or quantify the benefits of incorporating reference transcriptome in mapping RNA-Seq reads. In this paper, we have studied the impact of reference transcriptome on mapping RNA-Seq reads, especially on junction ones. The same dataset were analysed with and without RefGene transcriptome, respectively. Then a Perl script was developed to analyse and compare the mapping results. It was found that about 50–55% junction reads can be mapped to the same genomic regions regardless of the usage of RefGene model. More than one-third of reads fail to be mapped without the help of a reference transcriptome. For “Alternatively” mapped reads, i.e., those reads mapped differently with and without RefGene model, the mappings without RefGene model are usually worse than their corresponding alignments with RefGene model. For junction reads that span more than two exons, it is less likely to align them correctly without the assistance of reference transcriptome. As the sequencing technology evolves, the read length is becoming longer and longer. When reads become longer, they are more likely to span multiple exons, and thus the mapping of long junction reads is actually becoming more and more challenging without the assistance of reference transcriptome. Therefore, the advantages of using reference transcriptome in the mapping demonstrated in this study are becoming more evident for longer reads. In addition, the effect of the completeness of reference transcriptome on mapping of RNA-Seq reads is discussed.
doi:10.1371/journal.pone.0101374
PMCID: PMC4081564  PMID: 24992027

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