Accuracy and quality of massively parallel DNA pyrosequencing.
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.


CONCLUSIONS
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.
Huse
Susan M
SM
Josephine Bay Paul Center, Marine Biological Laboratory at Woods Hole, MBL Street, Woods Hole, MA 02543, USA.
Huber
Julie A
JA
Morrison
Hilary G
HG
Sogin
Mitchell L
ML
Welch
David Mark
DM
D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics; D014644:Genetic Variation; D005827:Genetics, Microbial / Q000379:methods*; D016678:Genome / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D004275:DNA, Ribosomal