Agilent sureselect human all exon v5 kit

High quality DNA was isolated using the Promega Wizard Genomic DNA Purification Kit (Promega, Wisconsin, United States) and the QIAamp DNA FFPE Tissue kit (Qiagen, Venlo, Netherlands) as previously described. One microgram of genomic DNA was used to produce exome-captured sequencing libraries using the Agilent SureSelect Human All Exon v5 kit (Agilent Technologies, California, United States). Paired-end 100-bp sequencing of each exome capture library was done using an Illumina HiSeq 2500 instrument and Illumina’s TruSeq SBS v3 chemistry (Illumina, California, United States).
Reads from tumor and matched normal blood sample were aligned separately to the human NCBI Build GRCh37 reference genome using Novoalign (Novocraft Technologies, Selangor, Malaysia) with default parameters. PCR duplicates, improper pairs and ambiguously mapped reads were removed using in-house scripts. SNVs were called using MuTect [13 (link), 14 (link)]. Variants annotation was done using Oncotator.

Exomes of the patient and his parents were captured from the genomic DNA using the Agilent SureSelect Human All Exon V5 kit (Agilent Technologies, Santa Clara, CA, USA) and were sequenced (paired-end, 2 × 124 bp) using the Illumina HiSeq 2500 (Illumina, San Diego, CA, USA). Read alignment to the human reference genome hg19 was performed with Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/), and ANNOVER was used for annotation. Germline mutations were detected through our established pipeline, as previously reported (http://genomon.hgc.jp/exome/en/index.html)^{19 (link)}. Population frequencies in the Human Genetic Variation Database (HGVD), db single-nucleotide polymorphisms (SNP) 131, the Integrative Japanese Genome Variation Database (iJGVD)²⁰ , and an in-house SNP database were used to filter common SNPs and to confirm the novelty of each mutation. Suspected pathogenic variations in genes known to be associated with ALF or present in accordance with autosomal recessive or X-linked modes of inheritance were identified as candidate genes.

WES sequencing libraries were prepare following the previous report with slight modifications^{72 (link)}.
In brief, genomic DNA was extracted from snap freezing GCA tumor or matched normal tissue using DNeasy Blood & Tissue Kit (69504, QIAGEN) according to the manufacturer’s instruction. DNA degradation and contamination were monitored on 1% agarose gels. DNA concentration was measured by Qubit DNA Assay Kit in Qubit 2.0 Flurometer (Invitrogen). A total amount of 0.6 μg genomic DNA per sample was fragmented to an average size of 180–280 bp and subjected to DNA library preparation using Illumina TruSeq DNA sample preparation kit. The Agilent SureSelect Human All ExonV5 Kit (5190-6209, Agilent Technologies) was used for exome capture according to the manufacturer’s instruction. In brief, DNA libraries were hybridized with liquid phase with biotin labeled probes from the Agilent SureSelect Human All ExonV5 Kit, then magnetic streptavidin beads were used to capture the exons of genes. Captured DNA fragments were enriched in a PCR reaction with index barcodes for sequencing. Final libraries were purified using AMPure XP beads (A63880, Beckman Coulter) and quantified using the Agilent high sensitivity DNA kit (5067-4626, Agilent Technologies). WES libraries were sequenced on Illumina Novaseq 6000 (Illumina) with 150 bp paired end mode according to the manufacturer instruction.

In all index cases, libraries of genomic DNA samples were prepared using the Agilent Sureselect Human All Exon v5 kit (Agilent Technologies, Santa Clara, CA, USA), and were sequenced on a HiSeq instrument (Illumina, San Diego, CA, USA) according to the manufacturer’s recommendations. Raw data were processed by NextGENe for alignment (SofGenetics, State College, PA, USA). The alignment, variant filtration and interpretation process is similar to these of Clinical exome sequencing. The average coverage depth was about 80–100×. Sequence variants were annotated using population and literature databases, including GnomAD, Clinvar, OMIM, and others. Classification of variants was performed with reference to the guideline recommended by the ACMG [18 (link)]. Sanger sequencing was used to verify the suspected mutation sites in fetus and parents.

Genomic DNA was extracted from the peripheral blood of the patient according to standard procedures using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, United States) and then sheared to 150 bp fragments in length. Whole-exome capture using the Agilent SureSelect Human All Exon v5 Kit (Agilent Technologies, Santa Clara, CA, United States) and high-throughput sequencing by utilizing an Illumina HiSeq 4000 sequencer (Illumina, San Diego, CA, United States) were conducted.
Computationally efficient read preprocessing and quality control for high-throughput sequencing data sets were taken with adaption of a canonical pipeline (Wang et al., 2013 (link)). The Trim Galore program was used to remove low-quality reads and adapters. The filtered reads (Phred-scaled quality score ≥ 30 and read length ≥ 80 bp) were aligned to the human reference genome (GRCH37/hg19) with the Burrows-Wheeler Alignment Tool (BWA) pipeline. Picard was then utilized to realign the reads from the BAM files and label the duplicated reads. In addition, local realignment and map quality score recalibration were performed. All variants, including single-nucleotide variants (SNVs) and InDels, were called according to three incorporated GATK tools: RealignerTargetCreator, IndelRealigner, and BaseRecalibrator. De novo and biallelic mutations were identified using mirTrios (Li et al., 2015 (link)).