Library preparation was done according to the Agilent SureSelectQXT workflow, and approximately 25 ng of input DNA was required. DNA was fragmented and the fragments tagged with adaptors. Tagged fragments were then purified, and the desired size was selected using AMPure beads (Beckman Coulter, CA, USA). DNA quality and quantity of the amplicons were assessed using the Qubit fluorometer (Invitrogen by Thermo-Fisher Scientific, South Africa) and the Bioanalyser 2100 (Agilent technologies, CA, USA). The purified amplicons were hybridized to the custom designed capture library. Hybridized amplicons were captured on streptavidin-coated beads (Thermo Fisher Scientific Inc. MA, USA) then amplified using indexing primers. Indexed libraries were then purified and pooled together in equimolar amounts before sequencing. Pooled libraries (6–10 pM) were sequenced in low (8 samples) and higher throughout (24 samples) batches by utilizing the appropriate MiSeq reagent v2 kits (nano, micro, or standard kits) on the Illumina MiSeq Instrument following manufacturer protocols (Illumina, CA, USA).
Sureselectqxt
Manufactured by Agilent Technologies
Sourced in United States
The SureSelectQXT is a library preparation kit designed for next-generation sequencing (NGS) workflows. It provides a streamlined and automatable process for preparing DNA samples for sequencing, optimized for use with Agilent's SureSelect target enrichment technology.
Automatically generated - may contain errors
Lab products found in correlation
Miseq instrument, by Illumina (2 mentions)
Sureselect xt, by Agilent Technologies (2 mentions)
Miseq reporter software, by Illumina (2 mentions)
Miseq platform, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Streptavidin coated beads, by Thermo Fisher Scientific (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Thruplex kit, by Takara Bio (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Miseq sequencer, by Illumina (1 mentions)
Sureselectxt human all exon v5, by Agilent Technologies (1 mentions)
Seqcap ez human exome library, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Clc genomics workbench, by Qiagen (1 mentions)
11 protocols using sureselectqxt
1
SureSelect Library Preparation and Sequencing
2
Comparison of Library Preparation Kits
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A total of five library preparation kits were tested: SureSelect-XT (Agilent Technologies, Santa Clara, CA, USA), SureSelect-QXT (Agilent Technologies, Santa Clara, CA, USA), KAPA Hyper (Kapa Biosystems Inc., Massachusetts, USA), NEBNext Ultra (New England Biolabs, Inc., Massachusetts, USA), and ThruPLEX kit(Rubicon Genomics, Miami, USA). To make the library using all kits except for SureSelect-QXT, genomic DNA was fragmented to 150–200 bp by sonication using a Covaris S2 (7 min, 0.5% duty, intensity = 0.1, 50 cycles/burst; Covaris Inc.) followed by purification using a 1.8× volume of AMPure XP Beads (Beckman Coulter, Indiana, USA). The SureSelect-QXT protocol utilized an enzymatic fragment process instead of sonication. After the fragmentation process, end-repair, A-tailing, adapter ligation, and PCR reactions before target enrichment was performed, following the manufacturer’s recommended protocols. After each step, the purification step was performed with AMPure beads to remove short fragments such as adapter dimers. Different adapters were used for comparison of kits according to each manufacturer’s protocol. Pre-indexed adapters were utilized for multiplexing hybrid selection.
3
Comprehensive NGS Lysosomal Disorder Panel
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Mutation analysis was performed by using a custom-made NGS gene panel (SureSelectQXT, Agilent Technologies). The panel targets coding exons of 61 genes described to be involved in lysosomal disorders (±25 bases, according to the RefSeq database and assembly February 2009 [GRCh37/hg19]). The list of genes is available upon request. Prepared libraries were sequenced in a paired-end run (2 × 150 bp) on the MiSeq instrument (Illumina). Demultiplexing, adaptor trimming, and mapping of sequencing reads to the human reference sequence hg19, as well as subsequent indel realigning and variant calling, was performed using the MiSeq Reporter Software (Illumina). Annotation and filtering of variants were addressed with Variant Studio Analysis Software (Illumina). Variants of interest were filtered according to allele frequency, exonic/splice site location, and autosomal recessive or X-linked pattern of inheritance. Copy-number variation analysis, executed with CLC Cancer Research Workbench 2.0 (CLC Bio, QIAGEN), was used to detect larger deletions (>1 kb). Quality assessment was performed by using Q30 as pass filter cutoff for a Phred score of called variants, together with a minimum sequencing read depth of 20× for target regions. Statistics for the NGS panel are summarized in Supplemental Table 1 .
4
Targeted Enrichment and Sequencing Protocol
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Targeted enrichment and sample preparation was done with the Agilent® SureSelectXT (http://www.chem.agilent.com ) automation kits on a Bravo® (http://www.chem.agilent.com ) liquid handling system. The SureSelectXT kit provides library preparation and targeted enrichment in one kit. The amount of input genomic DNA used in our study was 3 μg but more recent protocols like the transposase-based SureSelectQXT (Agilent) require 50 ng only. At our online resource (see QXT folder of the sftp repository; http://www.ikmb.uni-kiel.de/resources/download-tools/software/hlassign ) we provide example data for eight samples processed with the SureSelectQXT protocol. The RNA bait length was 120 bp. Sequencing was performed on an Illumina HiSeq2000® (http://systems.illumina.com ) with 100 bp paired-end runs. For the targeted enrichment we chose a fragment size between 150 and 300 base pairs and pooled 48 samples per lane. The 48 sample pools provided on average 5,044,060 paired reads per sample (median 4,481,579). We observed an increased error rate for the samples with less read count. So the calling becomes more confident with the higher number of reads obtained. It is difficult to determine an exact lower threshold for high confidence, but taking Supplementary Figure S1 into account we would suggest at least 7 million reads (3.5 million paired reads) as a lower threshold.
5
Comprehensive Genetic Panel Validation
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DNA was extracted from peripheral blood samples by standard procedures. DNA for panel validation from CEPH 1463 family members was obtained from the Coriell Institute. For genome partition, we used custom SureSelect QXT liquid capture kits (Agilent Technologies) according to the instructions of the manufacturer. Sure Select QXT technology fragments genomic DNA by using a proprietary transposase and subsequently captures selected regions by hybridization of specific RNA probes labeled with biotin. Adaptor‐ and barcode‐tagged gene libraries were deep sequenced in a MiSeq sequencer (Illumina) running in 2x150 bp, paired‐end reading setup. All putative causal variants detected by panel analysis were subsequently verified by Sanger sequencing. For the verification of mutations in those genes of the panel with non‐processed pseudogenes, we performed a gene‐specific, long‐range PCR amplification to generate the template for Sanger sequencing (primer sequences and long‐range PCR conditions available upon request). Additional specific PCR tests were carried out to ascertain the existence of possible whole‐exon deletions whenever suggested by copy‐number variation (CNV) analysis performed with XHMM software (see below).
6
Targeted Next-Generation Sequencing for Inherited Retinal Diseases
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Sixty-three patients were sequenced with a gene panel version (PV1) that included 117 genes involved in a non-syndromic IRD, and their flanking intronic regions (±25 base pairs) (Rodríguez-Muñoz et al., 2020 (link)). Moreover, the panel of genes contained five intronic regions of ABCA4, OFD1, USH2A, CEP290, and PRPF31, in which pathogenic variants had been previously identified (Den Hollander et al., 2006 (link); Littink et al., 2010 (link); Vaché et al., 2012 (link); Webb et al., 2012 (link); Braun et al., 2013 (link); Supplementary Table 1 ).
The remaining 29 cases were analyzed with an updated version of the custom panel (PV2) that had 114 genes and all the deep-intronic variants described in the last few years in ABCA4 and USH2A (Vaché et al., 2012 (link); Braun et al., 2013 (link); Zernant et al., 2014 (link); Bauwens et al., 2015 (link), 2019 (link); Liquori et al., 2016 (link); Baux et al., 2017 (link); Fadaie et al., 2019 (link); Khan et al., 2019 (link); Sangermano et al., 2019 (link);Supplementary Table 2 ).
The patients’ libraries were prepared in accordance with the SureSelect QXT protocol (Agilent Technologies) and sequenced on a MiSeq platform (Illumina, San Diego, CA) in 300 cycles with 2 × 150 base pairs reads.
The remaining 29 cases were analyzed with an updated version of the custom panel (PV2) that had 114 genes and all the deep-intronic variants described in the last few years in ABCA4 and USH2A (Vaché et al., 2012 (link); Braun et al., 2013 (link); Zernant et al., 2014 (link); Bauwens et al., 2015 (link), 2019 (link); Liquori et al., 2016 (link); Baux et al., 2017 (link); Fadaie et al., 2019 (link); Khan et al., 2019 (link); Sangermano et al., 2019 (link);
The patients’ libraries were prepared in accordance with the SureSelect QXT protocol (Agilent Technologies) and sequenced on a MiSeq platform (Illumina, San Diego, CA) in 300 cycles with 2 × 150 base pairs reads.
7
Exome Sequencing of Japanese Peripheral Blood Cells
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We sequenced libraries generated from genomic DNA derived from peripheral blood mononuclear cells of Japanese descent. Each exome captured sequencing library was produced from one of four different technologies: Roche/NimbleGen’s SeqCap EZ Human Exome Library v3.0, Illumina’s Nextera Rapid Capture Exome (v1.2), Agilent’s SureSelect XT Human All Exon v5 and Agilent’s SureSelect QXT. Each exome capture platform method was performed according to the manufacturer’s instructions except for Illumina platform. For the Illumina platform, we used 100 ng of total input genomic DNA. The captured DNA was sequenced using the Illumina HiSeq2500 platform with paired-end reads of 161bp for insert libraries according to the manufacturer’s instructions. We deposited all DNA sequence data used in this study to the National Bioscience Database Center (NBDC) Human Database (http://humandbs.biosciencedbc.jp/ ).
8
Rapid Whole Exome Sequencing for Fetal Diagnosis
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For fetal samples, DNA was isolated from fetal material without culturing of cells, whereas DNA from the parental samples was isolated from blood obtained through venipuncture. DNA library preparation was performed using SureSelect QXT in combination with the Sure Select All Human Exon Kit (v5, Agilent), followed by 2x150bp paired‐end sequencing on a NextSeq500 (Illumina). Sequence coverage was 200 to 300×. Automated data analysis pipeline included rapid BWA mapping, GATK variant calling and custom‐made annotation. Parental and fetal DNA was sequenced simultaneously in 53 of 54 cases (trio‐based analysis) to favor interpretation of results. For the remaining case, paternal DNA was unavailable.
Prior to rWES, aneuploidies for trisomy 13, 18 and 21 and monosomy X were excluded in all cases by quantitative fluorescent polymerase chain reaction (QF‐PCR) using routine procedures.22 Additionally, CMA was performed prior to (n = 22, 41%), or in‐parallel with (n = 25, 46%) rWES.
Prior to rWES, aneuploidies for trisomy 13, 18 and 21 and monosomy X were excluded in all cases by quantitative fluorescent polymerase chain reaction (QF‐PCR) using routine procedures.
9
Targeted Sequencing of Cardiac Genetics
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DNA was extracted from peripheral blood lymphocytes of the two siblings and their parents and analysed by next-generation sequencing (NGS) using a customized (SureDesign, based on an Agilent SureSelectQXT) solution-capture enrichment strategy (Agilent, Santa Clara, CA, USA). Targeted sequencing of ERS- and Brugada syndrome (BrS)-related genes (GPD1L, SCN1B, KCNE3, SCN3B, KCNH2, RANGRF, KCNE1L, KCND3, HCN4, SLMAP, TRPM4, SCN2B, SCN10A, KCNJ8, CACNA1C, CACNB2, CACNA2D1, SCN5A, and ABCC9) was performed in the index patient. Target regions included coding exons (padding: ±25 bp). Sequencing was carried out on a MiSeq™- or a NextSeq™ 500-System (Illumina, San Diego, CA, USA). Bioinformatic analyses were performed using CLC Genomics Workbench (Qiagen, Venlo, the Netherlands) and variants were verified by conventional Sanger sequencing.
10
Targeted Sequencing of CCM Genes
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Hybridization capture-based target enrichment of genomic DNA samples for CCM1, CCM2, and CCM3 was performed using an Agilent SureSelectQXT custom enrichment kit (Panel ID: 3152261, Agilent Technologies, Santa Clara, CA, USA). Illumina sequencing was performed on an Illumina MiSeq platform with 2 × 150 cycles (Illumina, San Diego, CA, USA). Basecalling and alignment to the GRCh37 reference genome were performed with the MiSeq Reporter Software (version 2.6.2, Illumina). Data were inspected with IGV [23 (link)] (version 2.13.0).
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