Identification and correction of systematic error in high-throughput sequence data

Frazer Meacham,Meromit Singer,Lior Pachter,David Ik Martin,Joseph Dhahbi,Dario Boffelli

doi:10.1186/1471-2105-12-451

Frazer Meacham, Meromit Singer + Show 4 more

Open Access

https://doi.org/10.1186/1471-2105-12-451

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Abstract

BackgroundA feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. The implications of such errors are application specific, ranging from minor informatics nuisances to major problems affecting biological inferences. Recently developed "next-gen" sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. We describe a new type of systematic error that manifests as statistically unlikely accumulations of errors at specific genome (or transcriptome) locations.ResultsWe characterize and describe systematic errors using overlapping paired reads from high-coverage data. We show that such errors occur in approximately 1 in 1000 base pairs, and that they are highly replicable across experiments. We identify motifs that are frequent at systematic error sites, and describe a classifier that distinguishes heterozygous sites from systematic error. Our classifier is designed to accommodate data from experiments in which the allele frequencies at heterozygous sites are not necessarily 0.5 (such as in the case of RNA-Seq), and can be used with single-end datasets.ConclusionsSystematic errors can easily be mistaken for heterozygous sites in individuals, or for SNPs in population analyses. Systematic errors are particularly problematic in low coverage experiments, or in estimates of allele-specific expression from RNA-Seq data. Our characterization of systematic error has allowed us to develop a program, called SysCall, for identifying and correcting such errors. We conclude that correction of systematic errors is important to consider in the design and interpretation of high-throughput sequencing experiments.

Highlights

A feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads
Characterizing systematic errors Having annotated the set of 2,116 systematic errors, we looked for characteristic features that could be identified in any high throughput sequencing experiment
As we have shown, distinguishing between systematic errors and heterozygous sites can be greatly improved by taking additional evidence into account

Summary

Introduction

A feature common to all DNA sequencing technologies is the presence of base-call errors in the sequenced reads. Developed “next-gen” sequencing technologies have greatly reduced the cost of sequencing, but have been shown to be more error prone than previous technologies. Both position specific (depending on the location in the read) and sequence specific (depending on the sequence in the read) errors have been identified in Illumina and Life Technology sequencing platforms. Systematic error manifests as many individual base-call errors from separate sequence reads occurring at the same genomic position (Figure 1). A systematic error comprises many individual base-call errors (from different reads) that fall at the same genomic location

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