Paired end libraries

Genomic DNA (1 μg) was fragmented by sonication. Illumina paired-end libraries from genomic DNA samples were constructed (Lupski et al., 2013 (link)). Pre-capture libraries were pooled and hybridized in solution to the BCM-HGSC CORE exome capture design (Bainbridge et al., 2011 (link)) (52Mb; NimbleGen). Captured DNA fragments were sequenced on an Illumina HiSeq 2000 platform, producing 9–10 gigabase-pairs (Gb) of sequence for each personal genome and achieving an average of 95% of the targeted exome bases covered to an average depth of 20x or greater, with mean coverage of target bases of over 100x. Raw sequence reads were mapped and aligned to the GRCh37 (hg19) human genome reference assembly using the HGSC Mercury analysis pipeline (http://www.tinyurl.com/HGSC-Mercury/) (Reid et al., 2014 (link)) or the Baylor Genetics analysis pipeline (Liu et al., 2019 (link)).

WES was performed in DIC 2 and DIC 5 (Table 1) using germline DNA extracted from bone marrow fibroblasts with normal karyotype. All variations identified by WES were confirmed as somatic on matched tumor/normal samples and tested on the remaining three DIC cases by Sanger sequencing. Illumina paired-end libraries were generated according to manufacturer’s protocol (Illumina San Diego, CA). Image processing and basecall were performed using the Illumina Real Time Analysis Software. Paired whole-exome fastq data were aligned to the human reference genome (GRCh38/hg38) with the BWA-MEM algorithm (10 (link)). Duplicates were marked using Samblaster. Quality of the aligned reads, somatic variant calling and copy number analysis were performed through CEQer2 tool, as previously described (11 (link)). Variants were annotated using dbSNP142. Variants with Minor Allele Frequency < 0.01 or carrying a ‘Clinical’ dbSNP flag were further processed; the other variants were discarded from subsequent analyses. Filtered variants were exported as vcf files and used as input for Annovar (12 (link)) analysis/annotation.