Sureselect human all exon v6

Twenty-three cases (including singletons, duos, trios, and quads) from the Center for Rare Childhood Disorders at The Translational Genomics Research Institute (TGen) were included in the analysis [60 (link), 61 (link)].
WES or WGS sequencing was carried out on an Illumina HiSeq 2000, HiSeq 2500, HiSeq 4000, or NovaSeq6000. For WES, the Agilent SureSelect Human All Exon V6 or CRE V2 target capture method was applied. Reads were aligned to reference GRCh37 version hs37d5 and variants called using GATK Haplotype caller version 3.3-0-g37228af (Broad Institute, Cambridge, MA).

Whole exome capture was performed using SureSelect Human All Exon V6 (Agilent Technologies). In brief, about 100 ng genomic DNA from each of the 65 paired tumor samples and 16 matched blood samples were extracted and fragmented using Covaris M220 (150 bp to 200 bp fragment size). The fragmented DNA was end-repaired and extended with an ‘A’ nucleotide at the 3’end. After PCR amplification and purification, the libraries were constructed using Agilent SureSelect kit according to the manufacturer’s instructions. The qualified exome-captured libraries were sequenced using Illumina HiSeq X Ten at Shanghai KR Pharmtech, Inc., Ltd. Adapters and low-quality reads were removed using trimmomatic (version 0.33)^{40 (link)}. Then the clean reads were aligned to the human reference genome (hg19) with Burrows-Wheeler Aligner (BWA; version 0.7.10)^{41 (link)}. All aligned reads were sorted, de-duplicated using Picard (version 1.103) (https://broadinstitute.github.io/picard/). Alignment quality assessment, local realignment and base quality recalibration were performed using the Genome Analysis Toolkit (GATK; version 3.1)^{42 (link)}. Mapping rate, coverage rate and duplicate rate were calculated with qualimap (version .2.1)^{43 (link)}. Soft-clipped reads were removed to reduce the influence of DNA degradation of FFPE samples.

For whole-exome sequencing, genomic DNA was extracted, and 1 μg was sheared and used for the construction of a paired-end sequencing library as described in the protocol provided by Illumina. Enrichment of exonic sequences was then performed for each library using the SureSelect Human All Exon V6 (Agilent Technologies Inc., Santa Clara, CA, USA) following the manufacturer’s instructions. Libraries for whole-exome sequencing were sequenced with a NovaSeq 6000 (Illumina Inc., San Diego, CA, USA).

Whole-exome sequencing was supported by Novogene Technology Co., Ltd. An Agilent SureSelect Human All Exon V6 exome analysis (Agilent, USA) was performed according to the manufacturer’s recommendations [28 (link), 29 (link)]. Each capture library was sequenced on the Illumina NovaSeq 6000 system (Illumina, USA) for paired-end 150 bp reads after pooling. The quality assessment tests of raw data were performed using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then, adapter sequences and low-quality fragments were removed from the 3′-end and analyzed by Trim Galore software. The clean sequence fragments were mapped to the human reference genome by BWA software and labeled repeats by Picard tools. The latest Best Practice process of GATK3.4 was used for sample variant detection, and ANNOVAR software was used for gene-based, region-based and filter-based site annotation [30 (link)].