Exon capture for 35 samples was performed using SureSelect Human All Exon V5 (Agilent), 35 samples with xGen Exome Research Panel v1 (Integrated DNA Technologies)), 42 samples with SureSelect Human All Exon V5+UTR (untranslated region) (Agilent), 25 samples with SureSelect Human All Exon V6+UTR (Agilent) and 5 samples with a focused exome sequencing panel that covers all the genes described in OMIM. Sequencing was performed on an Illumina HiSeq X system to generate 2×150 bp sequence reads with 8–10 GB of data per sample.
Xgen exome research panel v1
Manufactured by Integrated DNA Technologies
Sourced in United States
The XGen Exome Research Panel v1.0 is a comprehensive exome capture solution that targets the protein-coding regions of the human genome. It provides broad coverage of the exome to enable researchers to efficiently identify genetic variants associated with human health and disease.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (6 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Hiseq 4000 platform, by Illumina (3 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (3 mentions)
Kapa hyper dna library prep kit, by Roche (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Novaseq 6000 series sequencer, by Illumina (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Tianamp blood dna kit, by Tiangen Biotech (2 mentions)
Novoseq 6000, by Illumina (2 mentions)
Ampure xp, by Beckman Coulter (2 mentions)
Kapa hyper prep kit, by Roche (2 mentions)
Hiseq x system, by Illumina (1 mentions)
Sureselect human all exon v5, by Agilent Technologies (1 mentions)
Sureselect human all exon v6 utr, by Agilent Technologies (1 mentions)
Sureselect human all exon v5 utr, by Agilent Technologies (1 mentions)
Hybridization and wash reagents kit, by Integrated DNA Technologies (1 mentions)
Qiaamp dna ffpe tissue kit, by Thermo Fisher Scientific (1 mentions)
34 protocols using xgen exome research panel v1
1
Exome Capture and Sequencing for Genetic Studies
2
Exome Sequencing for Rare Skeletal Disorders
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Genomic DNA was extracted from fetal amniocytes and peripheral blood leukocytes of the parents using the salt fractionation method (Magen, Guangdong, China). Exome capture was performed using xGen Exome Research panel v1 (Integrated DNA Technologies, Coralville, IA, USA), and sequencing was conducted on NovaSeq 6000 (Illumina, San Diego, CA). The sequences were aligned to a human reference sequence (NCBI Genome build GRCh37) using the Burrows–Wheeler Aligner. The Genome Analysis Toolkit pipeline was used to detect single‐nucleotide (SNPs) and indel polymorphisms. Prioritization was given to variants that were previously reported, considered LOF (nonsense, frameshift, or splice site mutations), or absent/rare in gnomAD. The candidate gene list was then narrowed down based on abnormal long bone morphology (Human Phenotype Ontology [HP]: 0011314). Variant interpretation was performed in accordance with the ACMG/AMP guidelines and ClinGen specifications (Richards et al., 2015 (link); Zhang et al., 2020 (link)).
3
FFPE DNA Extraction and Exome Sequencing
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Genomic DNAs from FFPE sections were extracted with a QIAamp DNA FFPE Tissue Kit and quantified by Qubit 3.0 using the dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, USA). Library preparations were performed with a KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, USA). Target enrichment was performed using the xGen Exome Research Panel v1.0 and Hybridization and Wash Reagents Kit (Integrated DNA Technology, Coralville, USA) according to the manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 4000 platform using PE150 sequencing chemistry (Illumina, San Diego, USA) to a mean coverage depth of 150× for tumor samples and 50× for matched normal control samples.
4
Optimized Exome Sequencing Protocol
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Libraries were constructed according to an optimized protocol developed by the UW Medicine Center for Precision Diagnostics and Northwest Clinical Genomics Laboratory. Sample‐specific fragment libraries were prepared using the KAPA Hyper Prep DNA library kit (KAPA Biosystems, Wilmington, MA, USA) and subsequently enriched with the xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Coralvilla, IA, USA). The standard off‐the‐shelf probe set was supplemented with baits to capture missing targets, and boost coverage of poorly covered targets. Paired‐end sequencing of the exome‐enriched libraries was performed on a HiSeq 4000 instrument (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.
5
Genetic Analysis of Inherited Retinal Disorders
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A comprehensive custom gene panel of 295 known and candidate genes or a 429 gene-targeted panel linked to IRDs was used for genetic analyses, as reported in previous studies (Seong et al., 2015 (link); Kim et al., 2019 (link); Kim et al., 2021 (link)). Targeted next-generation sequencing (Targeted NGS, Illumina NextSeq 550 system; San Diego, CA, USA) or whole exome sequencing (WES; Illumina NovaSeq 6000 system) were performed. Targeted NGS was performed using custom-designed RNA oligonucleotide probes and a target enrichment kit (Celemics, Seoul, South Korea). WES was performed using the xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., Coraville, IA, USA) and SureSelect Human All Exon v6 enrichment kit (Agilent Technologies, Santa Clara, CA, USA).
6
Exome Sequencing of DNA Samples
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DNA was extracted from lymphocytes and tumour biopsies using QIAamp DNA Blood Mini and FFPE Tissue Kits (Qiagen), respectively, and sheared using a focusedultrasonicator (Covaris). Library construction was performed using the KAPA Hyper DNA Library Prep Kit. Genomic DNA libraries were amplified with HotStart Phusion Polymerase for 4-7 cycles prior to hybridisation. Exome enrichment was conducted using the xGEN® Exome Research Panel v1.0 (Integrated DNA Technologies). Captured libraries were amplified purified by AMPure XP beads, and quantified by qPCR (Kapa). Sequencing was carried out with the HiSeq Sequencing System (Illumina) with 2 × 150-bp paired-end reads. Sequencing reads were cleaned with QC, and aligned with BWA v0.7.12 to human genome (hg19). Picard tool http://picard.sourceforge.net/) was applied to eliminate duplicate reads. Small insertions and deletions (indels) and single nucleotide variants (SNVs) were analysed by UnifiedGenotyper, and annotated using the dbSNP v144 (https://www.ncbi.nlm.nih.gov/SNP/). Copy number variations (CNVs) were determined using the ADTEx (http://adtex.sourceforge.net/).
7
Whole Exome Sequencing for Rare Genetic Variants
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Whole exome sequencing was performed for the proband of Family 4, who did not have any mutations detected with respect to WNT10A, EDA, PAX9, and MSX1. Whole exome sequences were enriched with xGen® Exome Research Panel v1.0 (Integrated DNA Technologies, Coralville, IA, USA). The enriched samples were sequenced with an Illumina HiSeq 2000 to generate 150 bp paired-end reads. Reads were aligned to the human reference genome hg19 using the Burrows–Wheeler Aligner [34 (link)]. Single Nucleotide Polymorphisms (SNPs) and Insertions and Deletions (InDels) were identified by the Genome Analysis Toolkit (GATK) and annotated by ANNOVAR [35 (link),36 (link)]. Variants of EDAR, EDARADD, AXIN2, WNT10B, LRP6, and LTBP3 genes were extracted and filtered with the criteria of “minor allele frequency (MAF) < 1%” and “exonic”. The candidate mutation of EDAR was verified with PCR, followed by Sanger sequencing. PCR was performed, and the PCR products were sequenced as described in Section 2.2 . The reference sequence for EDAR was NM_022336.3.
8
Whole Exome Sequencing Protocol
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A total of 2 mL fasting blood was collected from the proband and her parents between 08:00 and 10:00 hours into ethylenediaminetetraacetic acid-treated tubes. Genomic DNA was extracted using the Blood Genome Column Medium Extraction Kit (Kangweishiji, Beijing, China) according to the manufacturer’s instructions. For whole exome library construction, protein-coding exome enrichment was performed using xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., Coralville, IA, USA). This consists of 429,826 individually synthesized and quality-controlled probes, targeting a 39-Mb protein-coding region (19,396 genes) of the human genome and covering 51 Mb of end-to-end tiled probe space. High-throughput sequencing was performed by an Illumina NovaSeq 6000 series sequencer (PE150; Illumina, San Diego, CA, USA), and not less than 99% of the target sequence was sequenced.
9
Whole-Exome Sequencing Approach
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DNA libraries were subjected to whole-exome capture with xGen Exome Research Panel v1.0 (Integrated DNA Technologies), which spans a 39-Mb target region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled space. The captured samples were sequenced on an Illumina HiSeq X-TEN platform with a paired-end run of 2 × 150 bp. See also Supplementary Material .
10
Exome Sequencing of Frozen Tumors
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We extracted genomic DNA from frozen tumors and normal (blood) samples and prepared sequencing libraries using an established protocol [20 (link)] that included enrichment with the xGEN® Exome Research Panel v1.0 from Integrated DNA Technologies. Sequencing was performed on an Illumina HiSeq4000 at The Genomics Services Laboratory at Nationwide Children’s Hospital (Columbus, Ohio) and achieved a median depth of 100–286x.
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