Target re-sequencing of 9p21 region

We selected 1.29 Mb interval of chromosome 9 (chr9:21299764–22590271, hg19) as a target region for next-generation sequencing (S1 Fig). The method for selecting target region is described in S1 Fig. We used NimbleGen SeqCap EZ choice system as a target enrichment method (Roche Diagnostics). A DNA probe set complementary to the target region was designed by NimbleDesign (https://design.nimblegen.com).

Genomic DNA was prepared from 48 Japanese patients with endometriosis using QIAamp DNA Blood Maxi Kit (QIAGEN) according to the manufacturer’s protocol as previously described [30, 57]. For each sample, 1 μg of genomic DNA was sheared into fragments with a mode length of about 200 bp on the Covaris (Covaris). Sequencing libraries were constructed with the Illumina TruSeq DNA sample Preparation Kit with 12 different indexed adapters (Illumina). Then, the target enrichment was performed with NimbleGen SeqCap EZ choice system according to the standard protocol (Roche Diagnostics). The libraries were sequenced on four runs of the Illumina MiSeq platform with 2×150-bp paired-end module (Illumina).

The reads containing the Illumina adapter sequences were trimmed by using Trimmomatic version 0.32 [58]. After the quality control step for excluding or trimming low quality sequences, the sequence reads were aligned to human reference genome (hg19) via BWA version 0.7 [59]. The aligned reads were processed for removal of PCR duplicates and erroneous reads by Picard tools version 1.111, and for local realignment and base quality recalibration by GATK version 3.2.2 [60, 61]. SNVs and indels were detected with the HaplotypeCaller and VariantRecalibrator of the GATK version 3.2.2 [60, 61].