Seqcap ez choice system

Genomic DNA was extracted from the whole peripheral blood cells of the participants.7 (link) We performed targeted exon sequencing of ABCG2 with a pool and capture method described in a previous study.8 (link) Briefly, the extracted DNA was quantified using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) on FilterMax F5 Multi-Mode Microplate Readers (Molecular Devices, Sunnyvale, California, USA). Twenty nanogram of DNA was simultaneously fragmented and ligated with adapters using the SureSelect QXT Library Prep Kit (Agilent Technologies, Santa Clara, California, USA). The 96 fragmented libraries with distinct indexed adapters were pooled in equimolar amounts. Target enrichment was then performed using the SeqCap EZ choice system (Roche Diagnostics, Tokyo, Japan). A DNA probe set complementary to the target region was designed using NimbleDesign (https://design.nimblegen.com). The libraries were sequenced on an Illumina HiSeq 2500 platform in rapid run mode with 2×150 bp paired-end modules (Illumina).

DNA extracted from two individuals’ LCLs (GM06990, CEU, female and GM19239, YRI, male) was mixed at different proportions, choosing 100%:0%, 99.5%:0.5%, 95%:5%, 90%:10%, 75%:25%, and 50%:50% to mimic different levels of mosaicism. The DNA was sequenced following a capture protocol using the SeqCap EZ Choice system from Roche-NimbleGen. The list of targeted regions and probes used for the v1MAD-seq capture design can be found in our dbGaP submission. Knowing the genotype of both samples, we were able to extract sites that mimic the different types of mosaic aneuploidy. Specifically, to simulate mitotic aneuploidy, we first extracted loci with different genotypes in the two cell lines, 0/1 in CEU and 1/1 in YRI to mimic overrepresentation of the alternate allele, and 0/1 in CEU and 0/0 in YRI to mimic underrepresentation of the alternate allele. The two mixtures separated further when a higher proportion of YRI DNA was mixed with CEU DNA (Supplemental Table S1). Because these mixtures of DNA alter the distribution of alternate allele frequency without changing the actual copy number of chromosomes, we applied the model without the coverage module.