title: Amplicon sequencing of Individual K8101-49685s
design: Validation variants were randomly selected from sets of particularly controversial variants, indels and SNVs unique to GATK, indels and SNVs unique to SOAP, and variants (both SNVs and indels) shared by these two pipelines. PCR primers were designed using the software program Primer 3 (sourceforge.net), to produce amplicons (ranging in size from 100 to 200 bp) containing variants of interest in approximately the center of the amplicon. Primers were obtained in 96-well plate format, 10 µmol/L dilution each (Sigma-Aldrich, St Louis, MO, USA). All primers were first tested for PCR efficiency using a HAPMAP DNA sample (Catalog ID NA12864l Coriell Institute for Medical Research, Camden, NJ, USA) and DNA polymerase (LongAmp® Taq DNA Polymerase; New England Biolabs, Beverly, MA, USA). k8101-49685 genomic DNA was used as template for the validation experiment. After quality-control steps using agarose gel, the product was purified (ExoSAP-IT® reagentsl Affymetrix Inc., Santa Clara, CA, USA) and pooled. Final PCR products were quantified (Qubit® dsDNA BR Assay Kitl Invitrogen Corp., Carlsbad, CA, USA), then library construction for the sequencer platform (MiSeq Personal Sequencer; Illumina Inc.) was performed. Finally, before being loaded onto the MiSeq machine, the quality and quantity of the sample was verified using the Bioanalyzer (Agilent Technologies) and quantitative PCR (Kapa Biosystems Inc., Woburn, MA, USA).