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1.  BFAST: An Alignment Tool for Large Scale Genome Resequencing 
PLoS ONE  2009;4(11):e7767.
Background
The new generation of massively parallel DNA sequencers, combined with the challenge of whole human genome resequencing, result in the need for rapid and accurate alignment of billions of short DNA sequence reads to a large reference genome. Speed is obviously of great importance, but equally important is maintaining alignment accuracy of short reads, in the 25–100 base range, in the presence of errors and true biological variation.
Methodology
We introduce a new algorithm specifically optimized for this task, as well as a freely available implementation, BFAST, which can align data produced by any of current sequencing platforms, allows for user-customizable levels of speed and accuracy, supports paired end data, and provides for efficient parallel and multi-threaded computation on a computer cluster. The new method is based on creating flexible, efficient whole genome indexes to rapidly map reads to candidate alignment locations, with arbitrary multiple independent indexes allowed to achieve robustness against read errors and sequence variants. The final local alignment uses a Smith-Waterman method, with gaps to support the detection of small indels.
Conclusions
We compare BFAST to a selection of large-scale alignment tools - BLAT, MAQ, SHRiMP, and SOAP - in terms of both speed and accuracy, using simulated and real-world datasets. We show BFAST can achieve substantially greater sensitivity of alignment in the context of errors and true variants, especially insertions and deletions, and minimize false mappings, while maintaining adequate speed compared to other current methods. We show BFAST can align the amount of data needed to fully resequence a human genome, one billion reads, with high sensitivity and accuracy, on a modest computer cluster in less than 24 hours. BFAST is available at http://bfast.sourceforge.net.
doi:10.1371/journal.pone.0007767
PMCID: PMC2770639  PMID: 19907642
2.  Sensitive and fast mapping of di-base encoded reads 
Bioinformatics  2011;27(14):1915-1921.
Motivation: Discovering variation among high-throughput sequenced genomes relies on efficient and effective mapping of sequence reads. The speed, sensitivity and accuracy of read mapping are crucial to determining the full spectrum of single nucleotide variants (SNVs) as well as structural variants (SVs) in the donor genomes analyzed.
Results: We present drFAST, a read mapper designed for di-base encoded ‘color-space’ sequences generated with the AB SOLiD platform. drFAST is specially designed for better delineation of structural variants, including segmental duplications, and is able to return all possible map locations and underlying sequence variation of short reads within a user-specified distance threshold. We show that drFAST is more sensitive in comparison to all commonly used aligners such as Bowtie, BFAST and SHRiMP. drFAST is also faster than both BFAST and SHRiMP and achieves a mapping speed comparable to Bowtie.
Availability: The source code for drFAST is available at http://drfast.sourceforge.net
Contact: calkan@u.washington.edu
doi:10.1093/bioinformatics/btr303
PMCID: PMC3129524  PMID: 21586516
3.  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 http://svdetect.sourceforge.net/
Contact: svdetect@curie.fr
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq293
PMCID: PMC2905550  PMID: 20639544
4.  Discovery of Novel MicroRNAs in Female Reproductive Tract Using Next Generation Sequencing 
PLoS ONE  2010;5(3):e9637.
MicroRNAs (miRNAs) are small non-coding RNAs that mediate post-transcriptional gene silencing. Over 700 human miRNAs have currently been identified, many of which are mutated or de-regulated in diseases. Here we report the identification of novel miRNAs through deep sequencing the small RNAome (<30 nt) of over 100 tissues or cell lines derived from human female reproductive organs in both normal and disease states. These specimens include ovarian epithelium and ovarian cancer, endometrium and endometriomas, and uterine myometrium and uterine smooth muscle tumors. Sequence reads not aligning with known miRNAs were each mapped to the genome to extract flanking sequences. These extended sequence regions were folded in silico to identify RNA hairpins. Sequences demonstrating the ability to form a stem loop structure with low minimum free energy (<−25 kcal) and predicted Drosha and Dicer cut sites yielding a mature miRNA sequence matching the actual sequence were considered putative novel miRNAs. Additional confidence was achieved when putative novel hairpins assembled a collection of sequences highly similar to the putative mature miRNA but with heterogeneous 3′-ends. A confirmed novel miRNA fulfilled these criteria and had its “star” sequence in our collection. We found 7 distinct confirmed novel miRNAs, and 51 additional novel miRNAs that represented highly confident predictions but without detectable star sequences. Our novel miRNAs were detectable in multiple samples, but expressed at low levels and not specific to any one tissue or cell type. To date, this study represents the largest set of samples analyzed together to identify novel miRNAs.
doi:10.1371/journal.pone.0009637
PMCID: PMC2835764  PMID: 20224791
5.  Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes 
BMC Genomics  2012;13:719.
Background
It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements.
Results
We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator.
Conclusions
This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.
doi:10.1186/1471-2164-13-719
PMCID: PMC3548764  PMID: 23260012
Breast cancer; Chromosome aberrations; Genomics; Fusion genes
6.  SAMMate: a GUI tool for processing short read alignments in SAM/BAM format 
Background
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.
Results
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.
Conclusions
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 http://sammate.sourceforge.net.
doi:10.1186/1751-0473-6-2
PMCID: PMC3027120  PMID: 21232146
7.  Identification of medium-sized genomic deletions with low coverage, mate-paired restricted tags 
BMC Genomics  2013;14:51.
Background
Genomic deletions are known to be widespread in many species. Variant sequencing-based approaches for identifying deletions have been developed, but their powers to detect those deletions that affect medium-sized regions are limited when the sequencing coverage is low.
Results
We present a cost-effective method for identifying medium-sized deletions in genomic regions with low genomic coverage. Two mate-paired libraries were separately constructed from human cancerous tissue to generate paired short reads (ditags) from restriction fragments digested with a 4-base restriction enzyme. A total of 3 Gb of paired reads (1.0× genome size) was collected, and 175 deletions were inferred by identifying the ditags with disorder alignments to the reference genome sequence. Sanger sequencing results confirmed an overall detection accuracy of 95%. Good reproducibility was verified by the deletions that were detected by both libraries.
Conclusions
We provide an approach to accurately identify medium-sized deletions in large genomes with low sequence coverage. It can be applied in studies of comparative genomics and in the identification of germline and somatic variants.
doi:10.1186/1471-2164-14-51
PMCID: PMC3608957  PMID: 23347462
Medium-sized deletion; Restriction enzymes; Next generation sequencing; Structural variation
8.  Detection of splicing events and multiread locations from RNA-seq data based on a geometric-tail (GT) distribution of intron length 
BMC Bioinformatics  2011;12(Suppl 5):S2.
Background
RNA sequencing (RNA-seq) measures gene expression levels and permits splicing analysis. Many existing aligners are capable of mapping millions of sequencing reads onto a reference genome. For reads that can be mapped to multiple positions along the reference genome (multireads), these aligners may either randomly assign them to a location, or discard them altogether. Either way could bias downstream analyses. Meanwhile, challenges remain in the alignment of reads spanning across splice junctions. Existing splicing-aware aligners that rely on the read-count method in identifying junction sites are inevitably affected by sequencing depths.
Results
The distance between aligned positions of paired-end (PE) reads or two parts of a spliced read is dependent on the experiment protocol and gene structures. We here proposed a new method that employs an empirical geometric-tail (GT) distribution of intron lengths to make a rational choice in multireads selection and splice-sites detection, according to the aligned distances from PE and sliced reads.
Conclusions
GT models that combine sequence similarity from alignment, and together with the probability of length distribution, could accurately determine the location of both multireads and spliced reads.
doi:10.1186/1471-2105-12-S5-S2
PMCID: PMC3226252  PMID: 21988959
9.  Long-Range Massively Parallel Mate Pair Sequencing Detects Distinct Mutations and Similar Patterns of Structural Mutability in Two Breast Cancer Cell Lines 
Cancer genetics  2011;204(8):447-457.
Cancer genomes frequently undergo genomic instability resulting in accumulation of chromosomal rearrangement. To date, one of the main challenges has been to confidently and accurately identify these rearrangements using short-read massively parallel sequencing. We were able to improve cancer rearrangement detection by combining two distinct massively parallel sequencing strategies: fosmid-sized (36 Kilobases on average) and standard 5 Kilobase mate pair libraries. We applied this strategy to map rearrangements in two breast cancer cell lines, MCF7 and HCC1954. We detect and validate a total of 91 somatic rearrangements in MCF7 and 25 in HCC1954, including genomic alterations corresponding to previously reported transcript aberrations in these two cell lines. Each of the genomes contains two types of breakpoints – clustered and dispersed. In both cell lines, the dispersed breakpoints show enrichment for low copy repeats, while the clustered breakpoints associate with high-copy number amplifications. Comparing the two genomes, we observe highly similar structural mutational spectra affecting different sets of genes, pointing to similar histories of genomic instability against the background of very different gene network perturbations.
doi:10.1016/j.cancergen.2011.07.009
PMCID: PMC3185296  PMID: 21962895
fosmid ditag; massively parallel sequencing; gene fusion; copy number variation; genomic instability
10.  Reconstructing cancer genomes from paired-end sequencing data 
BMC Bioinformatics  2012;13(Suppl 6):S10.
Background
A cancer genome is derived from the germline genome through a series of somatic mutations. Somatic structural variants - including duplications, deletions, inversions, translocations, and other rearrangements - result in a cancer genome that is a scrambling of intervals, or "blocks" of the germline genome sequence. We present an efficient algorithm for reconstructing the block organization of a cancer genome from paired-end DNA sequencing data.
Results
By aligning paired reads from a cancer genome - and a matched germline genome, if available - to the human reference genome, we derive: (i) a partition of the reference genome into intervals; (ii) adjacencies between these intervals in the cancer genome; (iii) an estimated copy number for each interval. We formulate the Copy Number and Adjacency Genome Reconstruction Problem of determining the cancer genome as a sequence of the derived intervals that is consistent with the measured adjacencies and copy numbers. We design an efficient algorithm, called Paired-end Reconstruction of Genome Organization (PREGO), to solve this problem by reducing it to an optimization problem on an interval-adjacency graph constructed from the data. The solution to the optimization problem results in an Eulerian graph, containing an alternating Eulerian tour that corresponds to a cancer genome that is consistent with the sequencing data. We apply our algorithm to five ovarian cancer genomes that were sequenced as part of The Cancer Genome Atlas. We identify numerous rearrangements, or structural variants, in these genomes, analyze reciprocal vs. non-reciprocal rearrangements, and identify rearrangements consistent with known mechanisms of duplication such as tandem duplications and breakage/fusion/bridge (B/F/B) cycles.
Conclusions
We demonstrate that PREGO efficiently identifies complex and biologically relevant rearrangements in cancer genome sequencing data. An implementation of the PREGO algorithm is available at http://compbio.cs.brown.edu/software/.
doi:10.1186/1471-2105-13-S6-S10
PMCID: PMC3358655  PMID: 22537039
11.  Accurate Identification and Analysis of Human mRNA Isoforms Using Deep Long Read Sequencing 
G3: Genes|Genomes|Genetics  2013;3(3):387-397.
Precise identification of RNA-coding regions and transcriptomes of eukaryotes is a significant problem in biology. Currently, eukaryote transcriptomes are analyzed using deep short-read sequencing experiments of complementary DNAs. The resulting short-reads are then aligned against a genome and annotated junctions to infer biological meaning. Here we use long-read complementary DNA datasets for the analysis of a eukaryotic transcriptome and generate two large datasets in the human K562 and HeLa S3 cell lines. Both data sets comprised at least 4 million reads and had median read lengths greater than 500 bp. We show that annotation-independent alignments of these reads provide partial gene structures that are very much in-line with annotated gene structures, 15% of which have not been obtained in a previous de novo analysis of short reads. For long-noncoding RNAs (i.e., lncRNA) genes, however, we find an increased fraction of novel gene structures among our alignments. Other important aspects of transcriptome analysis, such as the description of cell type-specific splicing, can be performed in an accurate, reliable and completely annotation-free manner, making it ideal for the analysis of transcriptomes of newly sequenced genomes. Furthermore, we demonstrate that long read sequence can be assembled into full-length transcripts with considerable success. Our method is applicable to all long read sequencing technologies.
doi:10.1534/g3.112.004812
PMCID: PMC3583448  PMID: 23450794
RNA; Roche sequencing; human; splicing; transcriptome
12.  DELLY: structural variant discovery by integrated paired-end and split-read analysis 
Bioinformatics  2012;28(18):i333-i339.
Motivation: The discovery of genomic structural variants (SVs) at high sensitivity and specificity is an essential requirement for characterizing naturally occurring variation and for understanding pathological somatic rearrangements in personal genome sequencing data. Of particular interest are integrated methods that accurately identify simple and complex rearrangements in heterogeneous sequencing datasets at single-nucleotide resolution, as an optimal basis for investigating the formation mechanisms and functional consequences of SVs.
Results: We have developed an SV discovery method, called DELLY, that integrates short insert paired-ends, long-range mate-pairs and split-read alignments to accurately delineate genomic rearrangements at single-nucleotide resolution. DELLY is suitable for detecting copy-number variable deletion and tandem duplication events as well as balanced rearrangements such as inversions or reciprocal translocations. DELLY, thus, enables to ascertain the full spectrum of genomic rearrangements, including complex events. On simulated data, DELLY compares favorably to other SV prediction methods across a wide range of sequencing parameters. On real data, DELLY reliably uncovers SVs from the 1000 Genomes Project and cancer genomes, and validation experiments of randomly selected deletion loci show a high specificity.
Availability: DELLY is available at www.korbel.embl.de/software.html
Contact: tobias.rausch@embl.de
doi:10.1093/bioinformatics/bts378
PMCID: PMC3436805  PMID: 22962449
13.  High-resolution mapping of copy-number alterations with massively parallel sequencing 
Nature methods  2008;6(1):99-103.
Cancer results from somatic alterations in key genes, including point mutations, copy number alterations and structural rearrangements. A powerful way to discover cancer-causing genes is to identify genomic regions that show recurrent copy-number alterations (gains and losses) in tumor genomes. Recent advances in sequencing technologies suggest that massively parallel sequencing may provide a feasible alternative to DNA microarrays for detecting copy-number alterations. Here, we present: (i) a statistical analysis of the power to detect copy-number alterations of a given size; (ii) SegSeq, an algorithm to identify chromosomal breakpoints using massively parallel sequence data; and (iii) analysis of experimental data from three matched pairs of tumor and normal cell lines. We show that a collection of ∼14 million aligned sequence reads from human cell lines has comparable power to detect events as the current generation of DNA microarrays and has over two-fold better precision for localizing breakpoints (typically, to within ∼1 kb).
doi:10.1038/nmeth.1276
PMCID: PMC2630795  PMID: 19043412
14.  GenomeView: a next-generation genome browser 
Nucleic Acids Research  2011;40(2):e12.
Due to ongoing advances in sequencing technologies, billions of nucleotide sequences are now produced on a daily basis. A major challenge is to visualize these data for further downstream analysis. To this end, we present GenomeView, a stand-alone genome browser specifically designed to visualize and manipulate a multitude of genomics data. GenomeView enables users to dynamically browse high volumes of aligned short-read data, with dynamic navigation and semantic zooming, from the whole genome level to the single nucleotide. At the same time, the tool enables visualization of whole genome alignments of dozens of genomes relative to a reference sequence. GenomeView is unique in its capability to interactively handle huge data sets consisting of tens of aligned genomes, thousands of annotation features and millions of mapped short reads both as viewer and editor. GenomeView is freely available as an open source software package.
doi:10.1093/nar/gkr995
PMCID: PMC3258165  PMID: 22102585
15.  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
16.  AGE: defining breakpoints of genomic structural variants at single-nucleotide resolution, through optimal alignments with gap excision 
Bioinformatics  2011;27(5):595-603.
Motivation: Defining the precise location of structural variations (SVs) at single-nucleotide breakpoint resolution is an important problem, as it is a prerequisite for classifying SVs, evaluating their functional impact and reconstructing personal genome sequences. Given approximate breakpoint locations and a bridging assembly or split read, the problem essentially reduces to finding a correct sequence alignment. Classical algorithms for alignment and their generalizations guarantee finding the optimal (in terms of scoring) global or local alignment of two sequences. However, they cannot generally be applied to finding the biologically correct alignment of genomic sequences containing SVs because of the need to simultaneously span the SV (e.g. make a large gap) and perform precise local alignments at the flanking ends.
Results: Here, we formulate the computations involved in this problem and describe a dynamic-programming algorithm for its solution. Specifically, our algorithm, called AGE for Alignment with Gap Excision, finds the optimal solution by simultaneously aligning the 5′ and 3′ ends of two given sequences and introducing a ‘large-gap jump’ between the local end alignments to maximize the total alignment score. We also describe extensions allowing the application of AGE to tandem duplications, inversions and complex events involving two large gaps. We develop a memory-efficient implementation of AGE (allowing application to long contigs) and make it available as a downloadable software package. Finally, we applied AGE for breakpoint determination and standardization in the 1000 Genomes Project by aligning locally assembled contigs to the human genome.
Availability and Implementation: AGE is freely available at http://sv.gersteinlab.org/age.
Contact: pi@gersteinlab.org
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq713
PMCID: PMC3042181  PMID: 21233167
17.  Multi-Sample Pooling and Illumina Genome Analyzer Sequencing Methods to Determine Gene Sequence Variation for Database Development 
Determination of sequence variation within a genetic locus to develop clinically relevant databases is critical for molecular assay design and clinical test interpretation, so multisample pooling for Illumina genome analyzer (GA) sequencing was investigated using the RET proto-oncogene as a model. Samples were Sanger-sequenced for RET exons 10, 11, and 13–16. Ten samples with 13 known unique variants (“singleton variants” within the pool) and seven common changes were amplified and then equimolar-pooled before sequencing on a single flow cell lane, generating 36 base reads. For comparison, a single “control” sample was run in a different lane. After alignment, a 24-base quality score-screening threshold and 3` read end trimming of three bases yielded low background error rates with a 27% decrease in aligned read coverage. Sequencing data were evaluated using an established variant detection method (percent variant reads), by the presented subtractive correction method, and with SNPSeeker software. In total, 41 variants (of which 23 were singleton variants) were detected in the 10 pool data, which included all Sanger-identified variants. The 23 singleton variants were detected near the expected 5% allele frequency (average 5.17%±0.90% variant reads), well above the highest background error (1.25%). Based on background error rates, read coverage, simulated 30, 40, and 50 sample pool data, expected singleton allele frequencies within pools, and variant detection methods; ≥30 samples (which demonstrated a minimum 1% variant reads for singletons) could be pooled to reliably detect singleton variants by GA sequencing.
PMCID: PMC2922832  PMID: 20808642
next generation sequencing; Illumina; RET; massively parallel sequencing
18.  Using paired-end sequences to optimise parameters for alignment of sequence reads against related genomes 
BMC Genomics  2010;11:458.
Background
The advent of cheap high through-put sequencing methods has facilitated low coverage skims of a large number of organisms. To maximise the utility of the sequences, assembly into contigs and then ordering of those contigs is required. Whilst sequences can be assembled into contigs de novo, using assembled genomes of closely related organisms as a framework can considerably aid the process. However, the preferred search programs and parameters that will optimise the sensitivity and specificity of the alignments between the sequence reads and the framework genome(s) are not necessarily obvious. Here we demonstrate a process that uses paired-end sequence reads to choose an optimal program and alignment parameters.
Results
Unlike two single fragment reads, in paired-end sequence reads, such as BAC-end sequences, the two sequences in the pair have a known positional relationship in the original genome. This provides an additional level of confidence over match scores and e-values in the accuracy of the positional assignment of the reads in the comparative genome. Three commonly used sequence alignment programs: MegaBLAST, Blastz and PatternHunter were used to align a set of ovine BAC-end sequences against the equine genome assembly. A range of different search parameters, with a particular focus on contiguous and discontiguous seeds, were used for each program. The number of reads with a hit and the number of read pairs with hits for the two end sequences in the tail-to-tail paired-end configuration were plotted relative to the theoretical maximum expected curve. Of the programs tested, MegaBLAST with short contiguous seed lengths (word size 8-11) performed best in this particular task. In addition the data also provides estimates of the false positive and false negative rates, which can be used to determine the appropriate values of additional parameters, such as score cut-off, to balance sensitivity and specificity. To determine whether the approach also worked for the alignment of shorter reads, the first 240 bases of each BAC end sequence were also aligned to the equine genome. Again, contiguous MegaBLAST performed the best in optimising the sensitivity and specificity with which sheep BAC end reads map to the equine and bovine genomes.
Conclusions
Paired-end reads, such as BAC-end sequences, provide an efficient mechanism to optimise sequence alignment parameters, for example for comparative genome assemblies, by providing an objective standard to evaluate performance.
doi:10.1186/1471-2164-11-458
PMCID: PMC3091654  PMID: 20678236
19.  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.
doi:10.1371/journal.pone.0058815
PMCID: PMC3608647  PMID: 23555596
20.  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
21.  Fast and SNP-tolerant detection of complex variants and splicing in short reads 
Bioinformatics  2010;26(7):873-881.
Motivation: Next-generation sequencing captures sequence differences in reads relative to a reference genome or transcriptome, including splicing events and complex variants involving multiple mismatches and long indels. We present computational methods for fast detection of complex variants and splicing in short reads, based on a successively constrained search process of merging and filtering position lists from a genomic index. Our methods are implemented in GSNAP (Genomic Short-read Nucleotide Alignment Program), which can align both single- and paired-end reads as short as 14 nt and of arbitrarily long length. It can detect short- and long-distance splicing, including interchromosomal splicing, in individual reads, using probabilistic models or a database of known splice sites. Our program also permits SNP-tolerant alignment to a reference space of all possible combinations of major and minor alleles, and can align reads from bisulfite-treated DNA for the study of methylation state.
Results: In comparison testing, GSNAP has speeds comparable to existing programs, especially in reads of ≥70 nt and is fastest in detecting complex variants with four or more mismatches or insertions of 1–9 nt and deletions of 1–30 nt. Although SNP tolerance does not increase alignment yield substantially, it affects alignment results in 7–8% of transcriptional reads, typically by revealing alternate genomic mappings for a read. Simulations of bisulfite-converted DNA show a decrease in identifying genomic positions uniquely in 6% of 36 nt reads and 3% of 70 nt reads.
Availability: Source code in C and utility programs in Perl are freely available for download as part of the GMAP package at http://share.gene.com/gmap.
Contact: twu@gene.com
doi:10.1093/bioinformatics/btq057
PMCID: PMC2844994  PMID: 20147302
22.  HeurAA: Accurate and Fast Detection of Genetic Variations with a Novel Heuristic Amplicon Aligner Program for Next Generation Sequencing 
PLoS ONE  2013;8(1):e54294.
Next generation sequencing (NGS) of PCR amplicons is a standard approach to detect genetic variations in personalized medicine such as cancer diagnostics. Computer programs used in the NGS community often miss insertions and deletions (indels) that constitute a large part of known human mutations. We have developed HeurAA, an open source, heuristic amplicon aligner program. We tested the program on simulated datasets as well as experimental data from multiplex sequencing of 40 amplicons in 12 oncogenes collected on a 454 Genome Sequencer from lung cancer cell lines. We found that HeurAA can accurately detect all indels, and is more than an order of magnitude faster than previous programs. HeurAA can compare reads and reference sequences up to several thousand base pairs in length, and it can evaluate data from complex mixtures containing reads of different gene-segments from different samples. HeurAA is written in C and Perl for Linux operating systems, the code and the documentation are available for research applications at http://sourceforge.net/projects/heuraa/
doi:10.1371/journal.pone.0054294
PMCID: PMC3548894  PMID: 23349847
23.  Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing 
Scientific Reports  2013;3:1186.
Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.
doi:10.1038/srep01186
PMCID: PMC3592332  PMID: 23470464
24.  Genome Size and Transposable Element Content as Determined by High-Throughput Sequencing in Maize and Zea luxurians 
The genome of maize (Zea mays ssp. mays) consists mostly of transposable elements (TEs) and varies in size among lines. This variation extends to other species in the genus Zea: although maize and Zea luxurians diverged only ∼140,000 years ago, their genomes differ in size by ∼50%. We used paired-end Illumina sequencing to evaluate the potential contribution of TEs to the genome size difference between these two species. We aligned the reads both to a filtered gene set and to an exemplar database of unique repeats representing 1,514 TE families; ∼85% of reads mapped against TE repeats in both species. The relative contribution of TE families to the B73 genome was highly correlated with previous estimates, suggesting that reliable estimates of TE content can be obtained from short high-throughput sequencing reads, even at low coverage. Because we used paired-end reads, we could assess whether a TE was near a gene by determining if one paired read mapped to a TE and the second read mapped to a gene. Using this method, Class 2 DNA elements were found significantly more often in genic regions than Class 1 RNA elements, but Class 1 elements were found more often near other TEs. Overall, we found that both Class 1 and 2 TE families account for ∼70% of the genome size difference between B73 and luxurians. Interestingly, the relative abundance of TE families was conserved between species (r = 0.97), suggesting genome-wide control of TE content rather than family-specific effects.
doi:10.1093/gbe/evr008
PMCID: PMC3068001  PMID: 21296765
maize; transposable elements; genome size; effective population size
25.  Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination 
Nucleic Acids Research  2011;40(3):e24.
Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3–5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome.
doi:10.1093/nar/gkr1000
PMCID: PMC3273786  PMID: 22127871

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