Accuracy and quality of massively parallel DNA pyrosequencing.

Abstract:

BACKGROUND:Massively parallel pyrosequencing systems have increased the efficiency of DNA sequencing, although the published per-base accuracy of a Roche GS20 is only 96%. In genome projects, highly redundant consensus assemblies can compensate for sequencing errors. In contrast, studies of microbial diversity that catalogue differences between PCR amplicons of ribosomal RNA genes (rDNA) or other conserved gene families cannot take advantage of consensus assemblies to detect and minimize incorrect base calls. RESULTS:We performed an empirical study of the per-base error rate for the Roche GS20 system using sequences of the V6 hypervariable region from cloned microbial ribosomal DNA (tag sequencing). We calculated a 99.5% accuracy rate in unassembled sequences, and identified several factors that can be used to remove a small percentage of low-quality reads, improving the accuracy to 99.75% or better. CONCLUSION:By using objective criteria to eliminate low quality data, the quality of individual GS20 sequence reads in molecular ecological applications can surpass the accuracy of traditional capillary methods.

journal_name

Genome Biol

journal_title

Genome biology

authors

Huse SM,Huber JA,Morrison HG,Sogin ML,Welch DM

doi

10.1186/gb-2007-8-7-r143

subject

Has Abstract

pub_date

2007-01-01 00:00:00

pages

R143

issue

7

eissn

1474-7596

issn

1474-760X

pii

gb-2007-8-7-r143

journal_volume

8

pub_type

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