For WES, libraries for sequencing on a HiSeq2000 instrument (Illumina Inc., USA) were prepared from genomic DNA and exome sequences enriched with SureSelect Human All Exon 50 M (Agilent Technologies, USA) according to the manufacturers’ instructions. The bioinformatic analysis of the raw data (paired-end reads) has been described previously [12 (link)]. In addition, the variants with the allele frequency > 1% in gnomAD database were excluded (https://gnomad.broadinstitute.org/ ). After a series of filtering steps, 40 sites in 38 genes (Additional file 1 : Table S1) were selected for further analysis. Both strands of the selected regions were sequenced by Sanger sequencing following polymerase chain reaction (PCR) amplification on an ABI 3730 Genetic Analyzer (Applied Biosystems Inc., USA). The results of Sanger sequencing were analyzed and presented using Chromas software (https://technelysium.com.au/wp/chromas/ ).
Sureselect human all exon 50 m
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect Human All Exon 50 M is a capture-based target enrichment system designed for whole exome sequencing. It targets the protein-coding regions of the human genome, covering approximately 50 Mb of the human exome.
Automatically generated - may contain errors
Lab products found in correlation
Sureselect human all exon v4, by Agilent Technologies (2 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Abi 3730 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Agilent rna 6000 nano labchip kit, by Agilent Technologies (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
3 protocols using sureselect human all exon 50 m
1
Whole Exome Sequencing and Variant Analysis
2
Whole Exome Sequencing of Blood and Nail Samples
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A total of 13 blood samples and paired normal fingernail samples were subjected to WES. In order to enrich the coding regions as much as possible, Sure Select Human All Exon 50M (Agilent Technologies, Inc.) was used for blood samples and Sure Select Human All Exon V4 (Agilent Technologies, Inc.) for nail samples. High-throughput sequencing of blood and nail samples was performed using an Illumina HiSeq2500 instrument (Illumina, Inc.). The average sequencing depths for blood and nail samples were 100× (range, 47–107×) and 102× (range, 96–106×), respectively. The sequencing depth of the above genes is presented in Fig. 1A . Broadband Wireless Access (BWA) was used to perform the alignment with the default parameters and human genome 19/the Genome Reference Consortium Human Genome Build 37 (hg19/GRCh37; ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens ) served as the reference genome. The relevant details were as follows: Genome version number, hg19/GRCh37; genome size, 3095677412 bp; capture chip, SeqCap EZ Human Exome Library NimbleGen v2.0; exon sequencing genes >20,000 genes; and capture target area size, 44.1 Mb. The associated 1000G database was utilized to annotate and report mutations in MAF (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ ).
3
Whole Exome and RNA Sequencing of Tumor Samples
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We performed WES on 10 tumor samples (S1–5 and F1–5) and eight paired normal blood samples (NS1–4 and NF1–4). To enrich the coding regions, we used SureSelect Human All Exon 50M (Agilent Technologies) for tumors and SureSelect Human All Exon V4 for normal samples. Sequence reads were produced using an Illumina HiSeq2000 instrument with a median on-target depth of 53× for tumor samples (range 47–77×) and 102× (range 96–106×) for normal samples (Supplementary Table S2 ). We performed the alignment using BWA [53 (link)] with the default parameters and hg19 as the reference genome.
RNA-seq was also performed on samples from the 13 tumors (S1–7 and F1–6). Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit) were used to determine the concentration and purity/integrity of RNA samples using an Agilent 2100 bioanalyzer. The sequencing libraries were prepared as previously described [54 (link)]. Raw reads from the Illumina HiSeq 2000 were aligned to the human reference genome (hg19) using the STAR [55 (link)] 2-pass method. Alignment performance was assessed using RNA-SeqQC (Supplementary Table S3 ), [56 (link)] and two samples (F3-4) with low throughput (# of mapped reads <10 Mb) were excluded from downstream analysis.
RNA-seq was also performed on samples from the 13 tumors (S1–7 and F1–6). Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit) were used to determine the concentration and purity/integrity of RNA samples using an Agilent 2100 bioanalyzer. The sequencing libraries were prepared as previously described [54 (link)]. Raw reads from the Illumina HiSeq 2000 were aligned to the human reference genome (hg19) using the STAR [55 (link)] 2-pass method. Alignment performance was assessed using RNA-SeqQC (
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