Dataset Information

Choice of reference-guided sequence assembler and SNP caller for analysis of Listeria monocytogenes short-read sequence data greatly influences rates of error.

ABSTRACT: The influences that different programs and conditions have on error rates of single-nucleotide polymorphism (SNP) analyses are poorly understood. Using Illumina short-read sequence data generated from Listeria monocytogenes strain HPB5622, we assessed the performance of four SNP callers (BCFtools, FreeBayes, UnifiedGenotyper, VarScan) under a variety of conditions, including: (1) a range of sequencing coverages; (2) use of four popular reference-guided assemblers (Burrows-Wheeler Aligner, Novoalign, MOSAIK, SMALT); (3) with and without read quality trimming and filtering; and (4) use of different reference sequences.At 8-fold coverage the proportions of true positive calls ranged from 0.22 to 25.00 % when reads were aligned to a nearly identical reference (0.000096 % distant). Calls made when reads were aligned to a non-identical reference (0.85 % distant) were from 92.54 to 98.88 % accurate. At 79-fold coverage accuracies ranged from 3.95 to 20.00 % with the nearly identical reference and 93.80-98.75 % with the non-identical reference. Read preprocessing significantly changed the numbers of false positive calls made, from a 65.24 % decrease to a 54.55 % increase.The combinations of reference-guided sequence assemblers and SNP callers greatly influenced not only the numbers of true and false positive sites but also the proportions of true positive calls relative to the total numbers of calls made. Furthermore, the efficacy of different assembler and caller combinations changed dramatically with the different conditions tested. Researchers should consider whether identifying the greatest numbers of true positive sites, reducing the numbers of false positive calls, or achieving the highest accuracies are desired.

SUBMITTER: Pightling AW

PROVIDER: S-EPMC4672502 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Choice of reference-guided sequence assembler and SNP caller for analysis of Listeria monocytogenes short-read sequence data greatly influences rates of error.

Pightling Arthur W AW Petronella Nicholas N Pagotto Franco F

BMC research notes 20151208

<h4>Background</h4>The influences that different programs and conditions have on error rates of single-nucleotide polymorphism (SNP) analyses are poorly understood. Using Illumina short-read sequence data generated from Listeria monocytogenes strain HPB5622, we assessed the performance of four SNP callers (BCFtools, FreeBayes, UnifiedGenotyper, VarScan) under a variety of conditions, including: (1) a range of sequencing coverages; (2) use of four popular reference-guided assemblers (Burrows-Whee ...[more]

PMID: 26643440

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Project description:The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results.

Project description:BACKGROUND:The challenges when developing a good de novo transcriptome assembler include how to deal with read errors and sequence repeats. Almost all de novo assemblers utilize a de Bruijn graph, with which complexity grows linearly with data size while suffering from errors and repeats. Although one can correct the errors by inspecting the topological structure of the graph, this is not an easy task when there are too many branches. Two research directions are to improve either the graph reliability or the path search precision, and in this study, we focused on the former. RESULTS:We present TraRECo, a greedy approach to de novo assembly employing error-aware graph construction. In the proposed approach, we built contigs by direct read alignment within a distance margin and performed a junction search to construct splicing graphs. While doing so, a contig of length l was represented by a 4 × l matrix (called a consensus matrix), in which each element was the base count of the aligned reads so far. A representative sequence was obtained by taking the majority in each column of the consensus matrix to be used for further read alignment. Once the splicing graphs had been obtained, we used IsoLasso to find paths with a noticeable read depth. The experiments using real and simulated reads show that the method provided considerable improvement in sensitivity and moderately better performance when comparing sensitivity and precision. This was achieved by the error-aware graph construction using the consensus matrix, with which the reads having errors were made usable for the graph construction (otherwise, they might have been eventually discarded). This improved the quality of the coverage depth information used in the subsequent path search step and finally the reliability of the graph. CONCLUSIONS:De novo assembly is mainly used to explore undiscovered isoforms and must be able to represent as many reads as possible in an efficient way. In this sense, TraRECo provides us with a potential alternative for improving graph reliability even though the computational burden is much higher than the single k-mer in the de Bruijn graph approach.

Dataset Information

Choice of reference-guided sequence assembler and SNP caller for analysis of Listeria monocytogenes short-read sequence data greatly influences rates of error.

Publications

Choice of reference-guided sequence assembler and SNP caller for analysis of Listeria monocytogenes short-read sequence data greatly influences rates of error.

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