title: BisQC: an operational pipeline for multiplexed bisulfite sequencing.
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.
  
  
  CONCLUSIONS
  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.
authors.forename: Gary G
authors.initials: GG
authors.lastname: Chen
authors.forename: Alpha B
authors.initials: AB
authors.lastname: Diallo
authors.forename: Raphaël
authors.initials: R
authors.lastname: Poujol
authors.forename: Corina
authors.initials: C
authors.lastname: Nagy
authors.forename: Alfredo
authors.initials: A
authors.lastname: Staffa
authors.forename: Kathryn
authors.initials: K
authors.lastname: Vaillancourt
authors.forename: Pierre-Eric
authors.initials: PE
authors.lastname: Lutz
authors.forename: Vanessa K
authors.initials: VK
authors.lastname: Ota
authors.forename: Deborah C
authors.initials: DC
authors.lastname: Mash
authors.forename: Gustavo
authors.initials: G
authors.lastname: Turecki
authors.affiliation: Department of Psychiatry, McGill University, Douglas Hospital Research Institute, Montreal, Quebec, Canada. carl.ernst@mcgill.ca.
authors.forename: Carl
authors.initials: C
authors.lastname: Ernst
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D017931:DNA Primers; D013447:Sulfites; C042345:hydrogen sulfite
references.citation: BMC Genomics. 2013 Nov 10;14:774
references.pmid: 24206606
references.citation: J Biomed Biotechnol. 2012;2012:741542
references.pmid: 23193365
references.citation: Cell. 2013 May 23;153(5):1149-63
references.pmid: 23664763
references.citation: Nat Nanotechnol. 2013 Jun;8(6):452-8
references.pmid: 23644569
references.citation: BMC Genomics. 2013 Oct 05;14:685
references.pmid: 24094292
references.citation: Genome Biol. 2012 Oct 03;13(10):R92
references.pmid: 23034176
references.citation: Neurosci Biobehav Rev. 2005 May;29(3):421-30
references.pmid: 15820547
references.citation: Nat Protoc. 2011 Apr;6(4):468-81
references.pmid: 21412275
references.citation: Science. 2001 Aug 10;293(5532):1089-93
references.pmid: 11498579
references.citation: Genome Biol. 2012 Oct 03;13(10):R87
references.pmid: 23034086
references.citation: Bioinformatics. 2012 Feb 1;28(3):430-2
references.pmid: 22155871
references.citation: Nucleic Acids Res. 2005 Oct 13;33(18):5868-77
references.pmid: 16224102
references.citation: Nat Biotechnol. 2011 Jan;29(1):24-6
references.pmid: 21221095
references.citation: Methods. 2009 Jul;48(3):226-32
references.pmid: 19442738
references.citation: Science. 1974 Apr 5;184(4132):80-1
references.pmid: 4815287
references.citation: Nucleic Acids Res. 2013 Jan;41(2):738-45
references.pmid: 23180759
references.citation: FEBS Lett. 2011 Jul 7;585(13):2049-58
references.pmid: 21420958
references.citation: Nucleic Acids Res. 1994 Aug 11;22(15):2990-7
references.pmid: 8065911
references.citation: J Mol Biol. 1965 Dec;14(2):603-7
references.pmid: 5880870
references.citation: BMC Bioinformatics. 2009 Jul 27;10:232
references.pmid: 19635165
references.citation: Nat Biotechnol. 2010 Oct;28(10):1045-8
references.pmid: 20944595
references.citation: Nat Neurosci. 2011 Aug 28;14(10):1345-51
references.pmid: 21874013
references.citation: Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3799-803
references.pmid: 8170990
references.citation: Nat Methods. 2010 Feb;7(2):133-6
references.pmid: 20062050
references.citation: PLoS Genet. 2012 Jun;8(6):e1002750
references.pmid: 22761581
references.citation: J Mol Biol. 1973 Oct 15;80(1):155-75
references.pmid: 4361747
references.citation: Hum Mol Genet. 2013 Oct 15;22(R1):R1-6
references.pmid: 23900077
references.citation: Nat Rev Genet. 2001 Jan;2(1):21-32
references.pmid: 11253064
references.citation: Nucleic Acids Res. 2012 May;40(10):e79
references.pmid: 22344695
references.citation: Nat Methods. 2010 Jun;7(6):461-5
references.pmid: 20453866
references.citation: Bioinformatics. 2011 Jun 1;27(11):1571-2
references.pmid: 21493656
references.citation: Bioinformatics. 2009 Nov 1;25(21):2841-2
references.pmid: 19736251
references.citation: Nature. 2011 Dec 14;480(7378):490-5
references.pmid: 22170606