BisQC: an operational pipeline for multiplexed bisulfite sequencing

Abstract

BackgroundBisulfite 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.ResultsWe 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.ConclusionWe 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.

Molecular Convergence of Neurodevelopmental Disorders

“…We followed BisQC, the multiplexed bisulfite sequencing parameters that we developed. 13,14 In brief, we used 5 mg of genomic DNA extracted from FBCs to carry out the MspI (New England Biolabs) digestion at 37 C for 7 hr (20 units of enzyme per microgram of DNA). We used the QIAGEN EpiTect Fast 96 Bisulfite Kit to carry out the bisulfite conversion of adaptor-ligated libraries and then sequenced four indexed samples per lane of an Illumina HiSeq 2000 by using 50 bp single-end reads.…”

“…To do this analysis, we performed RRBS by using our established pipeline 13 with two shRNAs per KD cell line and four nontarget controls. For the analysis, we segregated the genome into 500 bp windows and assessed those windows with at least two CpGs, and that showed a significant difference between methylation frequencies in KD and nontarget controls (mean methylation differences were >2%).…”

Estimation of the methylation pattern distribution from deep sequencing data

“…The rates of conversion and sequencing error can be estimated by spiking-in non-methylated DNA of known sequence, or by looking at positions known in advance not to be methylated [ 16 ].…”

Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine

“…RRBS was performed as described previously [8, 9]. Briefly, genomic DNA was extracted from iPSC-NPCs (D28) and digested with MspI.…”

“…Although this method, theoretically, covers every CpG (~30 million) in the human genome, getting adequate coverage on each individual CpG site is a costly and formidable task. Another genome-wide DNA modification methodology, reduced representation bisulfite sequencing (RRBS) [3, 4], utilizes methylation-insensitive restriction enzyme digestion and size selection to reduce the amount of genetic material being sequenced. Unlike WGBS, RRBS tends to extensively focus on CpG-rich regions [5, 6].…”