Targeted DNA sequencing libraries were constructed using SureSelectQXT Reagent Kit (Agilent Technologies, Santa Clara, CA) with 50 ng of genomic DNA. Briefly, tumor DNA was enzymatically fragmented and tagged to generate adapter-tagged libraries. Biotin-labeled probes specific to the targeted regions of interest (ROI) via hybridization, and libraries were enriched for ROI using streptavidin beads, then amplified, dual-indexed, and pooled for sequencing; quality of the libraries were measured with 2200 TapeStation (Agilent) and quantified using Qubit 2.0 (ThermoFisher Scientific, Waltham, MA). Targeted RNA sequencing (Fusion Panel) libraries were prepared using Archer™ Universal RNA Reagent Kit v2 for Illumina with 150 ng of input RNA or total nucleic acid (TNA) (ArcherDX, Boulder, CO). Target-enriched library was generated by using a combination of gene-specific primers and universal adapters with minor modification to the manufacturer’s instruction (ArcherDX). Briefly, RNA was reverse-transcribed to generate cDNA and molecular barcode adapters were ligated to cDNA followed by two rounds of target-specific PCR. The library was quantified using KAPA Biosystems qPCR kit (KAPA Biosystems, Wilmington, MA). All libraries were sequenced on Illumina MiSeq or HiSeq platform with MiSeq v2 or HiSeq Rapid SBS v2 300 cycle reagent kit (Illumina, San Diego, CA).
Sureselect qxt reagent kit
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect QXT reagent kit is a laboratory product designed for use in genomic analysis workflows. It provides the necessary reagents and materials required for library preparation prior to sequencing. The kit's core function is to enable the selective enrichment of targeted genomic regions of interest, facilitating efficient and accurate analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (2 mentions)
Miseq v2, by Illumina (1 mentions)
Kapa biosystems qpcr kit, by Roche (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Nextgene, by SoftGenetics (1 mentions)
Bcl2fastq software, by Illumina (1 mentions)
Qubit 4, by Thermo Fisher Scientific (1 mentions)
4200 bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Labchip gx touch nucleic acid analyzer, by PerkinElmer (1 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sureselectxt human all exon v5, by Agilent Technologies (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Abi prism 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
10 protocols using sureselect qxt reagent kit
1
Targeted DNA and RNA Sequencing Library Preparation
2
Comprehensive Solid Tumor Panel Sequencing
3
HER2 Copy Number Variation in Tissues and ctDNA
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The copy number variation of the HER2 gene in the gDNA of all tissue samples and ctDNA of blood samples was detected by NGS. The gDNA with a double terminal 8-base UDI connector and ctDNA with a single terminal 8-base plus 8-base UMI internal connector was used for the library construction, respectively. Then, the library was hybridized with an Agilent SureSelectQXT reagent kit and blocking agent (Agilent Technologies, Santa Clara, CA, USA). Finally, PCR was used to enrich the captured target bands, and the library was prepared after the capture. After fragment size quantitation by Qubit 4.0 (Thermo Fisher, Waltham, MA, USA) concentration and 4200 Bioanalyzer (Agilent), the library was quantified by quantitative polymerase chain reaction (qPCR), mixed, and then sequenced on NovaSeq 6000 platform (Illumina, San Diego, CA, USA; 4 g tissue data, 15 g blood data). The original data obtained after sequencing was automatically converted into FASTQ data using bcl2fastq software (Illumina) for subsequent data analysis.
4
Targeted DNA Sequencing of Prostate Cancer
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Genomic DNA isolated from prostate cancer tissues was submitted to the University of Minnesota Genomics Center for DNA-seq library preparation and hybrid capture with a custom SureSelect (Agilent) bait library using the SureSelect QXT reagent kit (Agilent) as per the manufacturer's recommendations. The SureSelect bait library was created using the SureSelect DNA Standard Design Wizard (Agilent) with 5 × tiling density, moderately stringent masking and MaximizePerformance parameters selected (Agilent). Post-capture sequencing libraries were pooled and diluted to 10 pM for flow cell clustering and sequenced in 2 lanes of an Illumina HiSeq 2500 with 2 × 150 bp settings for CRPC metastases, or 1 lane of an Illumina HiSeq 2500 with 2 × 125 bp settings for localized prostate cancer.
5
Bone Marrow RNA Extraction and Sequencing
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Total RNAs from the marrow samples were extracted by TRIzol reagent (Invitrogen, USA). Nanodrop 2000 spectrophotometer (Wilmington, USA) was used to measure the quantity and quality of the extracted RNA. Reverse transcription was performed according to the manufacture’s instruction by PrimeScriptTM RT reagent Kit (Takara, Japan). One hundred nanograms of sheared cDNAs was subjected to library construction with MGIEasy universal DNA library kit (MGI, China), then followed by hybrid capture using SureSelectQXT Reagent kit (Agilent, USA). Library quality and concentration were assessed by LabChip® GX Touch™ nucleic acid analyzer (PerkinElmer, USA) and Qubit fluorometer 3.0 (Life Technologies, USA), respectively. The qualified libraries were sequenced with 2 × 100 bp paired-end reads on a MGISEQ-2000 (MGI, China) platform.
6
Whole Corneal Exome Sequencing
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This pilot ES study was conducted with 50 ng of genomic DNA of whole corneal tissues (KC15, KC16, KC17, KC18, and KC19) using the SureSelectQXT Reagent Kit combined with the SureSelectXT Human All Exon V5 (Agilent Technologies, Cedar Creek, TX, USA) according to manufacturer’s instruction. Prepared libraries were paired-end sequenced (2 × 100 bp) on an Illumina HiSeq1500 (Illumina, San Diego, CA, USA). For each cornea sample > 80 million read pairs were generated resulted in >100× of mean coverage. Sequence readouts were initially analyzed with bcl2fastq software to generate reads in fastq format. These reads were mapped against a human genome reference sequence (GRCh37) using the Burrows–Wheeler Alignment (BWA), which was followed by BAM post-processing and variant calling using HaplotypeCaller and the GATK suite (McKenna et al., 2010 (link)). Finally, ANNOVAR (Wang, Li & Hakonarson, 2010 (link)) was used to annotate relevant information about gene names, predicted variant pathogenicity, reference allele frequencies and metadata from external resources and then to add these to the variant call format (VCF) file.
7
Low-pass WGS for Copy Number Analysis
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The QIAamp DNA mini kit (QIAGEN, Germantown, MD, USA) was used for genomic DNA (gDNA) extraction from bone marrow aspirate at diagnosis. A total of 50 ng of purified gDNA was fragmented to 600–1000 bp sizes and underwent adapter ligation, indexing, and amplification. The SureSelectQXT reagent kit (Agilent Technologies Inc., Santa Clara, CA, USA) was used for DNA library prep. Low-pass whole-genome sequencing (WGS) was performed as reported previously. Briefly, the mean target coverage was 8.86× with a mapping rate of 91.27%. Alignment to the GRCh38/hg38 reference genome was performed by using BWA13 and BWA-MEM, as reported previously [26 (link)]. The HMMcopy software (Bioconductor. DOI: 10.18129/B9.bioc.HMMcopy , (accessed on 20 June 2022)) [27 (link)] was applied to analyze the copy number status with focus on 2q, 3q, and 16q that were involved in translocations in our case.
8
Comprehensive Genetic Analysis of XPA and XPC
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This article is protected by copyright. All rights reserved ABI Prism 3130xl Genetic Analyzer (Applied Biosystems/Life Technologies). Primer sequences are detailed in Table S1. Data generated were analyzed using the CLC Main Workbench 5.0.2.
(CLC bio, Mühltal, Germany).
Nine negative cases for XPA and XPC pathogenic variants were evaluated by targeted nextgeneration sequencing (tNGS). One sample was evaluated exclusively by tNGS. DNA library preparation was performed using a custom SureSelectQXT reagent kit with DNA repair panel genes of 1.2Mb (Agilent Technologies, Santa Clara, CA, USA). The amplified libraries were sequenced (Illumina MiSeq system, San Diego, CA, USA) following the manufacturer's recommendations. The variant classification is described in Supplementary data.
To determine the haplotype of two distinct heterozygous variants, four of nine index cases had at least one of the parents tested for both variants. In one case whose parents were not available for testing, bidirectional allele-specific amplification was used (Table S2).
Alternative transcripts and protein expression analyses by immunohistochemistry were assessed in four cases to characterize XPC c.2251-1G>C (Supplementary data).
Co-segregation analysis was performed in symptomatic siblings from three families. Pedigrees were constructed using Progeny Software version 10.3.0.2 (Progeny Genetics, LLC).
(CLC bio, Mühltal, Germany).
Nine negative cases for XPA and XPC pathogenic variants were evaluated by targeted nextgeneration sequencing (tNGS). One sample was evaluated exclusively by tNGS. DNA library preparation was performed using a custom SureSelectQXT reagent kit with DNA repair panel genes of 1.2Mb (Agilent Technologies, Santa Clara, CA, USA). The amplified libraries were sequenced (Illumina MiSeq system, San Diego, CA, USA) following the manufacturer's recommendations. The variant classification is described in Supplementary data.
To determine the haplotype of two distinct heterozygous variants, four of nine index cases had at least one of the parents tested for both variants. In one case whose parents were not available for testing, bidirectional allele-specific amplification was used (Table S2).
Alternative transcripts and protein expression analyses by immunohistochemistry were assessed in four cases to characterize XPC c.2251-1G>C (Supplementary data).
Co-segregation analysis was performed in symptomatic siblings from three families. Pedigrees were constructed using Progeny Software version 10.3.0.2 (Progeny Genetics, LLC).
9
RNA-Seq Analysis of Ibrutinib-Treated Cell Lines
10
Whole Blood DNA Extraction and Targeted NGS
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Genomic DNA was isolated from 1 ml whole blood with EDTA as anticoagulant using the FlexiGene DNA Kit (Qiagen) according to the manufacturer's instructions. Tagmentation-based DNA library preparation and target gene enrichment were performed with the SureSelect QXT reagent kit (Agilent) and an Agilent SureSelect XT custom targets library (Agilent) according to the manufacturer's instructions. Paired-end short-read next generation sequencing was performed on an Illumina MiSeq DNA Sequencer Instrument using the Illumina MiSeq Reagent Kit v2. The bioinformatic analysis followed established best-practices using the Genome Analysis Toolkit (GATK) v.4.1.3.0. Briefly, this comprised sequence read adapter trimming (Cutadapt v.2.5, https://github.com/marcelm/cutadapt); sequence read alignment to the human reference genome version GRCh38 (bwa 0.7.17, http://bio-bwa.sourceforge.net); alignment processing and calling of sequence variants (GATK); and variant annotation (annovar v.2018-04-16, https://annovar.openbioinformatics.org).
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