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Challenges with using primer IDs to improve accuracy of next generation sequencing.

ABSTRACT: Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3?env plasmid clone and plasma samples from three HIV-infected patients. The Primer ID consisted of 11 randomized nucleotides, 4,194,304 combinations, in the primer for cDNA synthesis that introduced a unique sequence tag into each cDNA molecule. Consensus template sequences were constructed for reads with Primer IDs that were observed three or more times. Despite high numbers of input template molecules, the number of consensus template sequences was low. With 10,000 input molecules for the clone as few as 97 consensus template sequences were obtained due to highly skewed frequency of resampling. Furthermore, the number of sequenced templates was overestimated due to PCR errors in the Primer IDs. Finally, some consensus template sequences were erroneous due to hotspots for UDPS errors. The Primer ID methodology has the potential to provide highly accurate deep sequencing. However, it is important to be aware that there are remaining challenges with the methodology. In particular it is important to find ways to obtain a more even frequency of resampling of template molecules as well as to identify and remove artefactual consensus template sequences that have been generated by PCR errors in the Primer IDs.

SUBMITTER: Brodin J

PROVIDER: S-EPMC4351057 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Challenges with using primer IDs to improve accuracy of next generation sequencing.

Brodin Johanna J Hedskog Charlotte C Heddini Alexander A Benard Emmanuel E Neher Richard A RA Mild Mattias M Albert Jan J

PloS one 20150305 3

Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3Δenv plasmid ...[more]

PMID: 25741706

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Project description:To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.

Dataset Information

Challenges with using primer IDs to improve accuracy of next generation sequencing.

Publications

Challenges with using primer IDs to improve accuracy of next generation sequencing.

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