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Sureselectxt human all exon v5 capture library

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The SureSelectXT Human All Exon V5 capture library is a comprehensive genomic tool designed to target the protein-coding regions of the human genome, known as the exome. It provides a reliable and efficient method for selectively capturing and enriching the exonic sequences, allowing for in-depth analysis and identification of genetic variations within the coding regions.

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Lab products found in correlation

Hiseq 4000 sequencer, by Illumina (2 mentions) Hiseq 2000, by Illumina (2 mentions) Nebnext poly a mrna magnetic isolation module kit, by New England Biolabs (1 mentions) Nebnext ultra rna library prep kit for paired end multiplexed sequencing library, by Illumina (1 mentions) Rnalater rna stabilization reagent, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Sureselectxt target enrichment system for paired end sequencing library kit, by Illumina (1 mentions) Qiaamp dna blood midi kit, by Qiagen (1 mentions) Ion reporter, by Thermo Fisher Scientific (1 mentions) Clc genomics workbench 7, by Qiagen (1 mentions) Ion ampliseq custom panel, by Thermo Fisher Scientific (1 mentions)

4 protocols using sureselectxt human all exon v5 capture library

1

Genomic and Transcriptomic Profiling of Tumors

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Genomic DNA isolation and bulk DNA sequencing were performed as we described in our previous work [45 (link)]. Briefly, fresh tumors were surgically resected from these two patients. Each tissue was cut into two pieces, with one for further single-cell collection and the other for bulk sequencing. This procedure could maximally ensure that the single-cell and bulk sequencing data were generated from a close region of the tissue. Genomic DNA were extracted using the QIAamp DNA Mini Kit (QIAGEN). Exon libraries were constructed using the SureSelectXT Human All Exon V5 capture library (Agilent). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
For bulk RNA analysis, small fragments of tumor tissues were first stored in RNAlater RNA stabilization reagent (QIAGEN) after surgical resection and kept on ice to avoid RNA degradation. RNA of tumor samples were extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s specification. Libraries were constructed using NEBNext Poly (A) mRNA Magnetic Isolation Module kit (NEB) and NEBNext Ultra RNA Library Prep Kit for Illumina Paired-end Multiplexed Sequencing Library (NEB). Samples were sequenced on the Illumina Hiseq 4000 sequencer with 150-bp paired-end reads.
Liu F., Zhang Y., Zhang L., Li Z., Fang Q., Gao R, & Zhang Z. (2019). Systematic comparative analysis of single-nucleotide variant detection methods from single-cell RNA sequencing data. Genome Biology, 20, 242.
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2

Whole-Exome Sequencing of Tumor Samples

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Whole-exome sequencing was performed on genomic DNAs from two tumors and matched blood samples. The SureSelectXT Human All Exon V5 capture library (Agilent) for 50 Mb of exonic regions was used to capture the exonic DNA. According to the manufacturer’s instructions, we constructed the sequence library with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library kit (Agilent). Then DNA sequencing of 100-bp paired-end reads were performed using the Illumina HiSeq4000 sequencer.
Han X., Han Y., Tan Q., Huang Y., Yang J., Yang S., He X., Zhou S., Song Y., Pi J., Zuo L., Yao J., Wu D., Zhang Z, & Shi Y. (2019). Tracking longitudinal genetic changes of circulating tumor DNA (ctDNA) in advanced Lung adenocarcinoma treated with chemotherapy. Journal of Translational Medicine, 17, 339.
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3

Exome Sequencing of Parent-Patient Trio

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We performed WES in one parent-patient (Patient 1) trio because she had participated in another study of WES on brain malformation. To do this, genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Midi Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Genomic DNA was captured using the SureSelect XT Human All Exon V5 capture library (Agilent Technologies, Santa Clara, CA, USA), and sequenced using the Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) with 100 bp paired-end reads. Exome data processing, variant calling and variant annotation were performed as described previously [15 (link)].
Negishi Y., Miya F., Hattori A., Johmura Y., Nakagawa M., Ando N., Hori I., Togawa T., Aoyama K., Ohashi K., Fukumura S., Mizuno S., Umemura A., Kishimoto Y., Okamoto N., Kato M., Tsunoda T., Yamasaki M., Kanemura Y., Kosaki K., Nakanishi M, & Saitoh S. (2017). A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly. BMC Medical Genetics, 18, 4.
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4

Whole Exome Sequencing to Identify Genetic Variants

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Whole exome sequencing was performed on eight parent-patient trios (families 1–6). Genomic DNA was captured using the SureSelect XT Human All Exon V5 capture library (Agilent Technologies, Santa Clara, CA, USA), and sequenced using the Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) with 100 bp paired-end reads. Exome data processing, variant calling and variant annotation were performed as described previously35 (link), 36 (link). Multiplex targeted sequencing analysis was performed on one additional patient (patient 7.1). Amplicon libraries corresponding to the 44 exons of the EPG5 gene were prepared with an Ion AmpliSeq Custom Panel (Thermo Fisher Scientific, Waltham, MA, USA), and sequenced with an Ion Torrent Personal Genome Machine (PGM) system37 (link). Sequence data was analyzed using a CLC Genomics Workbench 7.0 (CLC bio, Aarhus, Denmark) and Ion reporter (Thermo Fisher Scientific). Identified mutations were validated in all nine patients by Sanger sequencing. The breakpoints in patient 7.1 were PCR amplified and sequenced. The ClinVar accession numbers for the DNA variants reported in this paper are SCV000328413, SCV000328414, SCV000328415, SCV000328416, SCV000328417, SCV000328418, and SCV000328419 (ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/).
Hori I., Otomo T., Nakashima M., Miya F., Negishi Y., Shiraishi H., Nonoda Y., Magara S., Tohyama J., Okamoto N., Kumagai T., Shimoda K., Yukitake Y., Kajikawa D., Morio T., Hattori A., Nakagawa M., Ando N., Nishino I., Kato M., Tsunoda T., Saitsu H., Kanemura Y., Yamasaki M., Kosaki K., Matsumoto N., Yoshimori T, & Saitoh S. (2017). Defects in autophagosome-lysosome fusion underlie Vici syndrome, a neurodevelopmental disorder with multisystem involvement. Scientific Reports, 7, 3552.
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