BisQC: an operational pipeline for multiplexed bisulfite sequencing
1 Department of Psychiatry, McGill University, Douglas Hospital Research Institute, Montreal, Quebec, Canada
2 McGill Group for Suicide Studies, Douglas Hospital Research Institute, 6875 LaSalle Boulevard, Frank Common Building, Room 2101.2 Verdun, Montreal, QC, H4H 1R3, Canada
3 McGill University and Genome Quebec Innovation Center, Montreal, Quebec, Canada
4 Departments of Neurology and Molecular and Cellular Pharmacology, Miller School of Medicine, Miami, Florida, USA
5 Department of Human Genetics, McGill University, Montreal, Quebec, Canada
BMC Genomics 2014, 15:290 doi:10.1186/1471-2164-15-290Published: 16 April 2014
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.
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.
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.