BisQC: an operational pipeline for multiplexed bisulfite sequencing.

TitleBisQC: an operational pipeline for multiplexed bisulfite sequencing.
Publication TypeJournal Article
Year of Publication2014
AuthorsChen GG, Diallo AB, Poujol R, Nagy C, Staffa A, Vaillancourt K, Lutz P-E, Ota VK, Mash DC, Turecki G, Ernst C
JournalBMC Genomics
Volume15
Pagination290
Date Published2014
ISSN1471-2164
KeywordsBase Sequence, DNA Primers, Polymerase Chain Reaction, Sequence Analysis, DNA, Sulfites
Abstract

BACKGROUND: Bisulfite sequencing is the most efficient single nucleotide resolution method for analysis of methylation status at whole genome scale, but improved quality control metrics are needed to better standardize experiments.RESULTS: We describe BisQC, a step-by-step method for multiplexed bisulfite-converted DNA library construction, pooling, spike-in content, and bioinformatics. We demonstrate technical improvements for library preparation and bioinformatic analyses that can be done in standard laboratories. We find that decoupling amplification of bisulfite converted (bis) DNA from the indexing reaction is an advantage, specifically in reducing total PCR cycle number and pre-selecting high quality bis-libraries. We also introduce a progressive PCR method for optimal library amplification and size-selection. At the sequencing stage, we thoroughly test the benefits of pooling non-bis DNA library with bis-libraries and find that BisSeq libraries can be pooled with a high proportion of non-bis DNA libraries with minimal impact on BisSeq output. For informatics analysis, we propose a series of optimization steps including the utilization of the mitochondrial genome as a QC standard, and we assess the validity of using duplicate reads for coverage statistics.CONCLUSION: We demonstrate several quality control checkpoints at the library preparation, pre-sequencing, post-sequencing, and post-alignment stages, which should prove useful in determining sample and processing quality. We also determine that including a significant portion of non-bisulfite converted DNA with bisulfite converted DNA has a minimal impact on usable bisulfite read output.

DOI10.1186/1471-2164-15-290
Alternate JournalBMC Genomics
PubMed ID24734894
PubMed Central IDPMC4234473
Grant ListDA033684 / DA / NIDA NIH HHS / United States

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