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1.  Seeing the Forest and the Trees: Visualizing Next-Generation Sequence Data 
The Generic Genome Browser (GBrowse) is an free, open source, web-based browser for displaying and navigating genome features.It is part of the Generic Model Organism Database (GMOD) project which aims to provide reusable components for working with genomic data, and is in use by hundreds of institutions around the world.GBrowse is relatively easy to install and can display any type of genome feature, including alignment data from current sequencing technologies after processing with SAMtools, a free and open source tool for working with large sequence alignments.The combination of SAMtools and GBrowse provides an excellent platform for providing extra value to sequencing core lab customers.See:,
PMCID: PMC2917996
2.  Bio-samtools: Ruby bindings for SAMtools, a library for accessing BAM files containing high-throughput sequence alignments 
The SAMtools utilities comprise a very useful and widely used suite of software for manipulating files and alignments in the SAM and BAM format, used in a wide range of genetic analyses. The SAMtools utilities are implemented in C and provide an API for programmatic access, to help make this functionality available to programmers wishing to develop in the high level Ruby language we have developed bio-samtools, a Ruby binding to the SAMtools library.
The utility of SAMtools is encapsulated in 3 main classes, Bio::DB::Sam, representing the alignment files and providing access to the data in them, Bio::DB::Alignment, representing the individual read alignments inside the files and Bio::DB::Pileup, representing the summarised nucleotides of reads over a single point in the nucleotide sequence to which the reads are aligned.
Bio-samtools is a flexible and easy to use interface that programmers of many levels of experience can use to access information in the popular and common SAM/BAM format.
PMCID: PMC3473260  PMID: 22640879
Next-generation sequencing; DNA; High; Throughput; Ruby; Bio; SAM; BAM
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.
PMCID: PMC3072548  PMID: 21320865
4.  BamView: viewing mapped read alignment data in the context of the reference sequence 
Bioinformatics  2010;26(5):676-677.
Summary: BamView is an interactive Java application for visualizing the large amounts of data stored for sequence reads which are aligned against a reference genome sequence. It supports the BAM (Binary Alignment/Map) format. It can be used in a number of contexts including SNP calling and structural annotation. BamView has also been integrated into Artemis so that the reads can be viewed in the context of the nucleotide sequence and genomic features.
Availability: BamView and Artemis are freely available (under a GPL licence) for download (for MacOSX, UNIX and Windows) at:
PMCID: PMC2828118  PMID: 20071372
5.  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:
PMCID: PMC3042176  PMID: 21208982
6.  SNP-o-matic 
Bioinformatics  2009;25(18):2434-2435.
Motivation: High throughput sequencing technologies generate large amounts of short reads. Mapping these to a reference sequence consumes large amounts of processing time and memory, and read mapping errors can lead to noisy or incorrect alignments. SNP-o-matic is a fast, memory-efficient and stringent read mapping tool offering a variety of analytical output functions, with an emphasis on genotyping.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2735664  PMID: 19574284
7.  Efficient digest of high-throughput sequencing data in a reproducible report 
BMC Bioinformatics  2013;14(Suppl 11):S3.
High-throughput sequencing (HTS) technologies are spearheading the accelerated development of biomedical research. Processing and summarizing the large amount of data generated by HTS presents a non-trivial challenge to bioinformatics. A commonly adopted standard is to store sequencing reads aligned to a reference genome in SAM (Sequence Alignment/Map) or BAM (Binary Alignment/Map) files. Quality control of SAM/BAM files is a critical checkpoint before downstream analysis. The goal of the current project is to facilitate and standardize this process.
We developed bamchop, a robust program to efficiently summarize key statistical metrics of HTS data stored in BAM files, and to visually present the results in a formatted report. The report documents information about various aspects of HTS data, such as sequencing quality, mapping to a reference genome, sequencing coverage, and base frequency. Bamchop uses the R language and Bioconductor packages to calculate statistical matrices and the Sweave utility and associated LaTeX markup for documentation. Bamchop's efficiency and robustness were tested on BAM files generated by local sequencing facilities and the 1000 Genomes Project. Source code, instruction and example reports of bamchop are freely available from
Bamchop enables biomedical researchers to quickly and rigorously evaluate HTS data by providing a convenient synopsis and user-friendly reports.
PMCID: PMC3846741  PMID: 24564231
8.  The variant call format and VCFtools 
Bioinformatics  2011;27(15):2156-2158.
Summary: The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API.
PMCID: PMC3137218  PMID: 21653522
9.  SAMMate: a GUI tool for processing short read alignments in SAM/BAM format 
Next Generation Sequencing (NGS) technology generates tens of millions of short reads for each DNA/RNA sample. A key step in NGS data analysis is the short read alignment of the generated sequences to a reference genome. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing this information.
We have developed a Graphical User Interface (GUI) software tool named SAMMate. SAMMate allows biomedical researchers to quickly process SAM/BAM files and is compatible with both single-end and paired-end sequencing technologies. SAMMate also automates some standard procedures in DNA-seq and RNA-seq data analysis. Using either standard or customized annotation files, SAMMate allows users to accurately calculate the short read coverage of genomic intervals. In particular, for RNA-seq data SAMMate can accurately calculate the gene expression abundance scores for customized genomic intervals using short reads originating from both exons and exon-exon junctions. Furthermore, SAMMate can quickly calculate a whole-genome signal map at base-wise resolution allowing researchers to solve an array of bioinformatics problems. Finally, SAMMate can export both a wiggle file for alignment visualization in the UCSC genome browser and an alignment statistics report. The biological impact of these features is demonstrated via several case studies that predict miRNA targets using short read alignment information files.
With just a few mouse clicks, SAMMate will provide biomedical researchers easy access to important alignment information stored in SAM/BAM files. Our software is constantly updated and will greatly facilitate the downstream analysis of NGS data. Both the source code and the GUI executable are freely available under the GNU General Public License at
PMCID: PMC3027120  PMID: 21232146
10.  Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology 
Bioinformatics  2010;26(14):1704-1707.
Motivation: The accuracy of reference genomes is important for downstream analysis but a low error rate requires expensive manual interrogation of the sequence. Here, we describe a novel algorithm (Iterative Correction of Reference Nucleotides) that iteratively aligns deep coverage of short sequencing reads to correct errors in reference genome sequences and evaluate their accuracy.
Results: Using Plasmodium falciparum (81% A + T content) as an extreme example, we show that the algorithm is highly accurate and corrects over 2000 errors in the reference sequence. We give examples of its application to numerous other eukaryotic and prokaryotic genomes and suggest additional applications.
Availability: The software is available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2894513  PMID: 20562415
11.  Development of Strategies for SNP Detection in RNA-Seq Data: Application to Lymphoblastoid Cell Lines and Evaluation Using 1000 Genomes Data 
PLoS ONE  2013;8(3):e58815.
Next-generation RNA sequencing (RNA-seq) maps and analyzes transcriptomes and generates data on sequence variation in expressed genes. There are few reported studies on analysis strategies to maximize the yield of quality RNA-seq SNP data. We evaluated the performance of different SNP-calling methods following alignment to both genome and transcriptome by applying them to RNA-seq data from a HapMap lymphoblastoid cell line sample and comparing results with sequence variation data from 1000 Genomes. We determined that the best method to achieve high specificity and sensitivity, and greatest number of SNP calls, is to remove duplicate sequence reads after alignment to the genome and to call SNPs using SAMtools. The accuracy of SNP calls is dependent on sequence coverage available. In terms of specificity, 89% of RNA-seq SNPs calls were true variants where coverage is >10X. In terms of sensitivity, at >10X coverage 92% of all expected SNPs in expressed exons could be detected. Overall, the results indicate that RNA-seq SNP data are a very useful by-product of sequence-based transcriptome analysis. If RNA-seq is applied to disease tissue samples and assuming that genes carrying mutations relevant to disease biology are being expressed, a very high proportion of these mutations can be detected.
PMCID: PMC3608647  PMID: 23555596
12.  SAMStat: monitoring biases in next generation sequencing data 
Bioinformatics  2010;27(1):130-131.
Motivation: The sequence alignment/map format (SAM) is a commonly used format to store the alignments between millions of short reads and a reference genome. Often certain positions within the reads are inherently more likely to contain errors due to the protocols used to prepare the samples. Such biases can have adverse effects on both mapping rate and accuracy. To understand the relationship between potential protocol biases and poor mapping we wrote SAMstat, a simple C program plotting nucleotide overrepresentation and other statistics in mapped and unmapped reads in a concise html page. Collecting such statistics also makes it easy to highlight problems in the data processing and enables non-experts to track data quality over time.
Results: We demonstrate that studying sequence features in mapped data can be used to identify biases particular to one sequencing protocol. Once identified, such biases can be considered in the downstream analysis or even be removed by read trimming or filtering techniques.
Availability: SAMStat is open source and freely available as a C program running on all Unix-compatible platforms. The source code is available from
PMCID: PMC3008642  PMID: 21088025
13.  Geno viewer, a SAM/BAM viewer tool 
Bioinformation  2012;8(2):107-109.
The ever evolving Next Generation Sequencing technology is calling for new and innovative ways of data processing and visualization. Following a detailed survey of the current needs of researchers and service providers, the authors have developed GenoViewer: a highly user-friendly, easy-to-operate SAM/BAM viewer and aligner tool. GenoViewer enables fast and efficient NGS assembly browsing, analysis and read mapping. It is highly customized, making it suitable for a wide range of NGS related tasks. Due to its relatively simple architecture, it is easy to add specialised visualization functionalities, facilitating further customised data analysis. The software's source code is freely available; it is open for project and task-specific modifications.
The database is available for free at
PMCID: PMC3282266  PMID: 22359445
14.  HUGO: Hierarchical mUlti-reference Genome cOmpression for aligned reads 
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.
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.
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 a General Public License (GPL) license.
Our method requires having different reference genomes and prolongs the execution time for additional alignments.
The proposed multi-reference-based compression algorithm for aligned reads outperforms existing single-reference based algorithms.
PMCID: PMC3932469  PMID: 24368726
15.  Bambino: a variant detector and alignment viewer for next-generation sequencing data in the SAM/BAM format 
Bioinformatics  2011;27(6):865-866.
Summary: Bambino is a variant detector and graphical alignment viewer for next-generation sequencing data in the SAM/BAM format, which is capable of pooling data from multiple source files. The variant detector takes advantage of SAM-specific annotations, and produces detailed output suitable for genotyping and identification of somatic mutations. The assembly viewer can display reads in the context of either a user-provided or automatically generated reference sequence, retrieve genome annotation features from a UCSC genome annotation database, display histograms of non-reference allele frequencies, and predict protein-coding changes caused by SNPs.
Availability: Bambino is written in platform-independent Java and available from, along with documentation and example data. Bambino may be launched online via Java Web Start or downloaded and run locally.
PMCID: PMC3051333  PMID: 21278191
16.  SVDetect: a tool to identify genomic structural variations from paired-end and mate-pair sequencing data 
Bioinformatics  2010;26(15):1895-1896.
Summary: We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions–deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization.
Availability: Source code and sample data are available at
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC2905550  PMID: 20639544
17.  SnoopCGH: software for visualizing comparative genomic hybridization data 
Bioinformatics  2009;25(20):2732-2733.
Summary: Array-based comparative genomic hybridization (CGH) technology is used to discover and validate genomic structural variation, including copy number variants, insertions, deletions and other structural variants (SVs). The visualization and summarization of the array CGH data outputs, potentially across many samples, is an important process in the identification and analysis of SVs. We have developed a software tool for SV analysis using data from array CGH technologies, which is also amenable to short-read sequence data.
Availability and implementation: SnoopCGH is written in java and is available from
PMCID: PMC2759554  PMID: 19687029
18.  ABMapper: a suffix array-based tool for multi-location searching and splice-junction mapping 
Bioinformatics  2010;27(3):421-422.
Summary: 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.
Availability and Implementation: The software is freely available at: The software is written in C++ and PERL. It runs on all major platforms and operating systems including Windows, Mac OS X and LINUX.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3031031  PMID: 21169377
19.  MethylCoder: software pipeline for bisulfite-treated sequences 
Bioinformatics  2011;27(17):2435-2436.
Motivation: MethylCoder is a software program that generates per-base methylation data given a set of bisulfite-treated reads. It provides the option to use either of two existing short-read aligners, each with different strengths. It accounts for soft-masked alignments and overlapping paired-end reads. MethylCoder outputs data in text and binary formats in addition to the final alignment in SAM format, so that common high-throughput sequencing tools can be used on the resulting output. It is more flexible than existing software and competitive in terms of speed and memory use.
Availability: MethylCoder requires only a python interpreter and a C compiler to run. Extensive documentation and the full source code are available under the MIT license at:
PMCID: PMC3157921  PMID: 21724594
20.  Searching for SNPs with cloud computing 
Genome Biology  2009;10(11):R134.
Novel software utilizing cloud computing technology to cost-effectively align and map SNPs from a human genome in three.
As DNA sequencing outpaces improvements in computer speed, there is a critical need to accelerate tasks like alignment and SNP calling. Crossbow is a cloud-computing software tool that combines the aligner Bowtie and the SNP caller SOAPsnp. Executing in parallel using Hadoop, Crossbow analyzes data comprising 38-fold coverage of the human genome in three hours using a 320-CPU cluster rented from a cloud computing service for about $85. Crossbow is available from
PMCID: PMC3091327  PMID: 19930550
21.  Variant Callers for Next-Generation Sequencing Data: A Comparison Study 
PLoS ONE  2013;8(9):e75619.
Next generation sequencing (NGS) has been leading the genetic study of human disease into an era of unprecedented productivity. Many bioinformatics pipelines have been developed to call variants from NGS data. The performance of these pipelines depends crucially on the variant caller used and on the calling strategies implemented. We studied the performance of four prevailing callers, SAMtools, GATK, glftools and Atlas2, using single-sample and multiple-sample variant-calling strategies. Using the same aligner, BWA, we built four single-sample and three multiple-sample calling pipelines and applied the pipelines to whole exome sequencing data taken from 20 individuals. We obtained genotypes generated by Illumina Infinium HumanExome v1.1 Beadchip for validation analysis and then used Sanger sequencing as a “gold-standard” method to resolve discrepancies for selected regions of high discordance. Finally, we compared the sensitivity of three of the single-sample calling pipelines using known simulated whole genome sequence data as a gold standard. Overall, for single-sample calling, the called variants were highly consistent across callers and the pairwise overlapping rate was about 0.9. Compared with other callers, GATK had the highest rediscovery rate (0.9969) and specificity (0.99996), and the Ti/Tv ratio out of GATK was closest to the expected value of 3.02. Multiple-sample calling increased the sensitivity. Results from the simulated data suggested that GATK outperformed SAMtools and glfSingle in sensitivity, especially for low coverage data. Further, for the selected discrepant regions evaluated by Sanger sequencing, variant genotypes called by exome sequencing versus the exome array were more accurate, although the average variant sensitivity and overall genotype consistency rate were as high as 95.87% and 99.82%, respectively. In conclusion, GATK showed several advantages over other variant callers for general purpose NGS analyses. The GATK pipelines we developed perform very well.
PMCID: PMC3785481  PMID: 24086590
22.  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:;
Requirements: Java 1.6 or higher.
PMCID: PMC3603209  PMID: 22253280
genome browser; next-generation sequencing; visualization; Artemis; BamView
23.  A random-permutations-based approach to fast read alignment 
BMC Bioinformatics  2013;14(Suppl 5):S8.
Read alignment is a computational bottleneck in some sequencing projects. Most of the existing software packages for read alignment are based on two algorithmic approaches: prefix-trees and hash-tables. We propose a new approach to read alignment using random permutations of strings.
We present a prototype implementation and experiments performed with simulated and real reads of human DNA. Our experiments indicate that this permutations-based prototype is several times faster than comparable programs for fast read alignment and that it aligns more reads correctly.
This approach may lead to improved speed, sensitivity, and accuracy in read alignment. The algorithm can also be used for specialized alignment applications and it can be extended to other related problems, such as assembly.
More information:
PMCID: PMC3622637  PMID: 23734846
24.  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
PMCID: PMC3792961  PMID: 24116042
25.  Compression of next-generation sequencing reads aided by highly efficient de novo assembly 
Nucleic Acids Research  2012;40(22):e171.
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
PMCID: PMC3526293  PMID: 22904078

Results 1-25 (374413)