The ethics committee of the Shanghai Children’s Medical Center approved the study, and informed consent was obtained from all participants. DNA was extracted from patients’ peripheral blood and sheared with a Covaris S2 Ultrasonicator (Covaris, Woburn, MA, USA) to create fragments of 150 to 200 bp. Sequencing library preparation and target capture were performed using the SureSelectXT Library Prep Kit and the SureSelectXT Human All Exon v4 Capture Library (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocol. Clusters were generated by isothermal bridge amplification with an Illumina cBot station, and sequencing was performed using the Illumina HiSeq 2000 System (Illumina, San Diego, CA, USA), with 100 base-pair read-lengths. Four samples were pooled into a single flow cell lane.
Sureselectxt human all exon v4 capture library
Manufactured by Agilent Technologies
Sourced in United States
The SureSelectXT Human All Exon v4 Capture Library is a targeted gene enrichment solution designed for sequencing the protein-coding regions of the human genome. It is a comprehensive kit that captures the exonic regions of the human genome, enabling efficient and cost-effective sequencing of the human exome.
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2 protocols using sureselectxt human all exon v4 capture library
1
Targeted DNA Sequencing of Patients
2
Exome Sequencing Protocol for HG19 Alignment
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Exome paired-end libraries were prepared using the Agilent (Chicago, IL) SureSelectXT Human All Exon V4 capture library. 2×100 bp reads were obtained using an Illumina (San Diego, CA) HiSeq 2500 instrument in Rapid Run mode on a HiSeq Rapid v1 flowcell. Indexed reads were de-multiplexed with CASAVA v1.8.2.
Paired-end sequencing reads were trimmed using fastX_Toolkit and aligned to HG19/GRCh37 human reference genome (http://www.genome.ucsc.edu ) using BWA-mem. The output was then sorted, indexed and PCR duplicates were removed using SAMTOOLS [24] (link). Bam files were then locally realigned and target loci marked using GATK IndelRealigner and TargetIntervals. MST alleles were retrieved and analyzed using software described in the next section.
Paired-end sequencing reads were trimmed using fastX_Toolkit and aligned to HG19/GRCh37 human reference genome (
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