Accuracy and quality of massively parallel DNA pyrosequencing

By Susan M. Huse, J. Huber, Hilary G. Morrison, Mitchell L Sogin, D. Welch. Genome Biology, 2007

1,285 citations recorded by the source corpus.

BackgroundMassively 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.ResultsWe 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.ConclusionBy 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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b1abf125ee210202d05d9e21df121531e0f6b759

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Citations in the corpus

Soil microbial community structure and diversity are largely influenced by soil pH and nutrient quality in 78-year-old tree plantations (2017, methodology): “The default filter settings were used: no ambiguous bases were allowed (Huse et al., 2007) and the minimum read length was 150 bp.”
FunGene: the functional gene pipeline and repository (2013, background): “The presence of Ns (uncalled bases) in primer regions has been linked to low quality sequences (Huse et al., 2007).”
Who is eating what: diet assessment using next generation sequencing (2012, background): “For example, 454 technology is known for its low reliability during homopolymer extension (Huse et al. 2007).”
Applications of next generation sequencing in molecular ecology of non-model organisms (2011, background): “However, 454 sequencing presents slightly higher rates of sequencing errors compared with competing technologies (see Table 1), especially in homopolymers (see Glossary) (Huse et al., 2007).”
DNA Barcoding for Honey Biodiversity (2010, background): “To limit the influence of sequence errors [31], only sequences that were present more than three times were considered in the subsequent analyses.”
Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform (2015, background): “The majority of errors in 454 data are related to homopolymers (1,2).”
Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies (2011, background): “Huse and colleagues [12] found that if they removed any read that had ambiguous base calls, mismatches to the PCR primer, or were shorter or longer than expected, then the observed error rate was reduced to 0.”
Metagenomic Analyses: Past and Future Trends (2010, background): “correction by assembly and sequencing depth, which is typically applied during genome projects, is not feasible (51, 64).”
Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels (2012, background): “5–5% [20,21,25,28] on Roche, Illumina, and Applied Biosystems platforms to 18% on the Pacific Biosciences platform [23].”
A polyphenol-rich cranberry extract protects from diet-induced obesity, insulin resistance and intestinal inflammation in association with increased Akkermansia spp. population in the gut microbiota of mice (2014, methodology): “This model allows choosing appropriate statistical methods to evaluate differences in the proportions of sequences assigned to different taxonomic groups between metagenomes, while considering effect sizes and confidence intervals in assessing biologically relevant differences.”
Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing (2011, background): “They also demonstrated that 39% of the errors corresponded to homopolymer effects [10].”
Rhizosphere-associated bacterial network structure and spatial distribution differ significantly from bulk soil in wheat crop fields (2017, background): “Reads which were 200bp long or had an average quality score 25 were removed (Huse et al., 2007), resulting in 5,595,603 high quality sequences.”
Removing Noise From Pyrosequenced Amplicons (2011, background): “It has previously been shown that some features correlate with noise in reads from 454 data [4,6].”
The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing (2008, methodology): “Pyrosequencing reads of both the V6 and V3 regions were passed through additional quality filters to reduce the overall error rate [43].”
Fast Identification and Removal of Sequence Contamination from Genomic and Metagenomic Datasets (2011, background): “therefore this analysis provides a worst-case scenario [38].”
Microbial Dysbiosis in Colorectal Cancer (CRC) Patients (2011, methodology): “Raw sequencing reads were quality trimmed according to published recommendations [31].”
Comparison of Next-Generation Sequencing Systems (2012, methodology): “An exemplary application of the Ion Torrent PGM sequencer is the identification of microbial pathogens.”
Characterization of the Diversity and Temporal Stability of Bacterial Communities in Human Milk (2011, background): “5% [36], these errors significantly inflate the number of observed OTUs [37].”