In total, 200 ng of genomic DNA was fragmented into 250‐ to 350‐bp fragments using sonication followed by end repair. The DNA fragments were purified and ligated with adapters using a SureSelectXT Library Prep Kit (Agilent, Santa Clara, CA). Next, the adapter‐ligated DNA libraries were amplified and captured using a SureSelect capture library kit (Agilent). The captured sequences were further amplified for 350‐bp read‐length paired‐end sequencing using an Illumina X‐ten system (Illumina, San Diego, CA).
Sureselectxt library prep kit
Manufactured by Agilent Technologies
Sourced in United States
The SureSelectXT Library Prep Kit is a core laboratory product designed for the preparation of sequencing libraries. It provides a streamlined workflow for the construction of libraries from DNA samples, enabling downstream sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (2 mentions)
Axen cancer panel 2, by Macrogen (2 mentions)
Nextseq 500 midoutput system platform, by Illumina (2 mentions)
X ten system, by Illumina (1 mentions)
Ovation ultralow system v2, by Tecan (1 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bcl2fastq2, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Novaseq, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Agencourt ampure xp reagent, by Beckman Coulter (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Sureselect human all exon v5, by Agilent Technologies (1 mentions)
Sureselect all exon v5 kit, by Agilent Technologies (1 mentions)
S2 ultrasonicator, by Agilent Technologies (1 mentions)
22 protocols using sureselectxt library prep kit
1
DNA Fragmentation and Library Preparation
2
Early-Onset Cerebellar Atrophy Genetic Analysis
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The family included in this study was referred to the departments of pediatric neurology and genetics of the Necker Enfants Malades Hospital. High-resolution karyotype, array-comparative genomic hybridization (aCGH) (Agilent 60 K) was performed. Informed consents have been obtained both from the participants and the legal representatives of the children. Sequencing was performed with a custom gene panel including 72 genes involved in early-onset cerebellar atrophy or PCH as previously described [32 (link)]. Briefly, genomic DNA libraries were generated using SureSelectXT Library PrepKit (Agilent) and the Ovation Ultralow System V2 (NuGen) according to the suppliers’ recommendations. All exons and 25 base pairs intronic flanking sequences of the 72 selected genes were captured by hybridization with biotinylated complementary 120-bp RNA baits designed with SureSelect SureDesign software. Paired-end sequences were mapped on the human reference genome (NCBI build37/ hg19 version) using the Burrows-Wheeler Aligner. Downstream processing was carried out with the Genome Analysis Toolkit (GATK), SAMtools, and Picard. Sanger sequencing was performed on the patient and parents’ blood samples using the 3500xL Genetic Analyzer (Applied Biosystems).
3
Targeted Sequencing for Variant Detection
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Coding and flanking intronic regions were enriched using a SureSelectXT Library Prep Kit (Agilent) and a CeGaT tumor panel (Agilent), and were sequenced using an Illumina NovaSeq6000 system (CeGaT GmbH Tübingen). Illumina bcl2fastq2 was used to demultiplex sequencing reads. Adapter removal was performed with Skewer. The trimmed reads were mapped to the human reference genome (hg19) using the Burrows Wheeler Aligner (BWA-mem Version 0.7.2-cegat) [28 (link)]. Local realignment of reads in target areas was performed using ABRA [29 (link)] to achieve more accurate indel calling. In reference hg19, the pseudoautosomal regions (PAR) on chromosome Y have been masked (chrY:10001–2649520, chrY:59034050–59363566). This prevents reads that fall within this homologous region on chromosome X and Y from being assigned to more than one chromosome and, thus, being discarded during mapping. Reads mapping to more than one location with an identical mapping score were discarded. Read duplicates that likely result from PCR amplification were removed using samtools (Version 0.1.18) [28 (link)]. The remaining high-quality sequences were used to determine sequence variants (single nucleotide changes and small insertions/deletions). The variants were annotated based on several internal as well as external databases and on information from scientific literature.
4
Whole Exome Sequencing for Monogenic Disease
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Because the majority of monogenic diseases can be detected in the coding part of the genome, the DNA samples of the two patients underwent WES using Illumina NOVASEQ6000 platform (Illumina Inc., San Diego, CA, USA). Exomes were captured using SureSelectXT Library Prep Kit (Agilent Technologies, USA). The sequencing reads (150 bp pair end) were mapped to the reference genome (UCSC hg19) using the Burrows-Wheeler Aligner software [5 (link)]. Polymerase chain reaction duplicates were removed using samblaster [6 (link)]. Single-nucleotide variants and small insertions/deletions (indels) were called using freebayes [7 ] and annotated using SnpEff-3.3 (Ensembl-GRCh37.73) [8 (link)]. Sequencing was conducted by Macrogen (Seoul, Republic of Korea) and the pipeline megSAP was used [9 ].
To identify possible disease-causing mutations, all high-quality variants were identified that are located in the protein coding region (according to Ensembl database v68) and/or two base pair flanking splice sites. We maintained only the variants meeting the following quality criteria: (1) at least 10X coverage and (2) mapping quality score ≥60.
To identify possible disease-causing mutations, all high-quality variants were identified that are located in the protein coding region (according to Ensembl database v68) and/or two base pair flanking splice sites. We maintained only the variants meeting the following quality criteria: (1) at least 10X coverage and (2) mapping quality score ≥60.
5
Genotyping Workflow for ClinPharmSeq
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To test the genotyping ability and investigate the potential limitations of ClinPharmSeq, we sequenced a total of 64 Coriell DNA samples of diverse ancestry (S1 Table ). Libraries were prepared for sequencing using the SureSelectXT Library Prep Kit from Agilent Technologies, Inc. per the manufacturer’s recommendations with minor modification. Sequencing was performed with the NextSeq 500 System from Illumina, Inc. (San Diego, CA, USA) using 150 base pairs (bp) paired-end reads. Raw sequencing data were produced in the Binary Base Call (BCL) file format, which were then converted to individual FASTQ files by demultiplexing with the bcl2fastq program (v2.19.0). We aligned sequence reads in the FASTQ files to the Human Genome version 19 (hg19) reference genome using the ‘ngs-fq2bam’ command from the fuc package (v0.26.0, https://github.com/sbslee/fuc ). The command generates a pipeline for automatically converting FASTQ files to ‘analysis-ready’ Binary Alignment Map (BAM) files by combining various commands from the BWA [28 (link)], SAMtools [29 (link)], and Genome Analysis Toolkit (GATK) [30 (link)] programs. Finally, we used the ‘bedcov’ command from SAMtools (v1.13) to calculate average coverage across the target space.
6
Whole Exome Sequencing and Variant Prioritization
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Three micrograms of genomic DNA were prepared for whole exome sequencing with
the Agilent SureSelect XT Library Prep Kit according to the manufacturer’s
protocol (Agilent Technologies). Sequencing was performed on an Illumina
HiSeq2500 with a 100-bp paired-end protocol. The FASTQ files were aligned to
the human reference genome (GRCh37) with the Burrows-Wheeler aligner (Li and Durbin
2009 ). The resulting alignment was processed according to Genome
Analysis Toolkit best practices (Van der Auwera et al. 2013 (link)).
Indel and single-nucleotide variants were called in the VCF format with the
Haplotype Caller function of the Genome Analysis Toolkit program. With the
VCFhacks package (https://github.com/gantzgraf/vcfhacks ), variants present in
the National Center for Biotechnology Information’s dbSNP147 or the Exome
Aggregation Consortium’s database (version 0.3) with a minor allele
frequency ≥0.1% were excluded. Remaining variants were annotated with the
National Center for Biotechnology Information’s Variant Effect Predictor.
Variants with a Combined Annotation Dependent Depletion (CADD; version 1.3)
score ≥15 were prioritized, and those in genes already known to cause AI
were highlighted for segregation analysis.
the Agilent SureSelect XT Library Prep Kit according to the manufacturer’s
protocol (Agilent Technologies). Sequencing was performed on an Illumina
HiSeq2500 with a 100-bp paired-end protocol. The FASTQ files were aligned to
the human reference genome (GRCh37) with the Burrows-Wheeler aligner (
2009
Analysis Toolkit best practices (Van der Auwera et al. 2013 (link)).
Indel and single-nucleotide variants were called in the VCF format with the
Haplotype Caller function of the Genome Analysis Toolkit program. With the
VCFhacks package (
the National Center for Biotechnology Information’s dbSNP147 or the Exome
Aggregation Consortium’s database (version 0.3) with a minor allele
frequency ≥0.1% were excluded. Remaining variants were annotated with the
National Center for Biotechnology Information’s Variant Effect Predictor.
Variants with a Combined Annotation Dependent Depletion (CADD; version 1.3)
score ≥15 were prioritized, and those in genes already known to cause AI
were highlighted for segregation analysis.
7
Capture-C Library Generation and Sequencing
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We generated colonoid Capture C libraries following an established protocol.23 ,24 (link) Each library then was sonicated using a QSonica (QSonica, Newtown, CT) Q800R to obtain DNA fragments with an average size of 350 bp. DNA fragments were purified using AMPureXP beads (Agencourt) and measured via Qubit (Invitrogen, Carlsbad, CA) fluorometer. Fragment quality and sizes were assessed on a Bioanalyzer (Agilent Technologies, Carlsbad, CA) 2100 using a 1000 DNA Chip. DNA ends were repaired and adaptors were ligated using the SureSelectXT Library Prep Kit (Agilent). After a clean-up step, the sizes and concentrations of DNA fragments were checked again. To generate high-complexity libraries, adaptor ligated DNA fragments were hybridized with a custom-designed capture panel (Agilent)24 (link) using the SureSelectXT capture kit (Agilent). Each captured library was first paired-end sequenced on 1-lane HiSeq 4000 sequencing (100-bp read length) for quality control and then sequenced on an S2 flow cells on an Illumina NovaSeq (50-bp read length). Data were analyzed as previously described.24 (link)
Promoter-focused Capture-C libraries from HNs were generated and analyzed previously in our laboratory.23
Promoter-focused Capture-C libraries from HNs were generated and analyzed previously in our laboratory.23
8
FFPE Tissue Exome Sequencing
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Genomic DNA was extracted from formalin‐fixed, paraffin embedded (FFPE) tissue sections using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Frankfurt, Germany), according to the manufacturer's protocol. DNA was assessed for quality and was qualified using NanoDrop and agarose gel electrophoresis. DNA libraries were created using the SureSelect XT Library Prep Kit (Agilent, Palo Alto, CA, USA), according to the manufacturer's protocol. Exome capture was performed with the Agilent SureSelect kit. The DNA library and exomes were sequenced on the Illumina HiSeq 2500 platform (Illunima, San Diego, CA, USA). The capture and coverage of raw reads were mapped to a human genome reference provided by the Burrows–Wheeler Aligner and reference sequence version HG19 alignment. Indels were called using gatk , and all variants were annotated by annovar .
9
Genomic Analysis of Cardiovascular Disorders
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Prior informed consent, a saliva sample of the proband was obtained and sent to Health in Code for processing in line with our in‐house protocol (https://www.ncbi.nlm.nih.gov/gtr/labs/320229/ ). After genomic DNA isolation, 10 ng of DNA was used for library construction, exon capture, and paired‐end read sequencing using the SureSelect XT Library Prep Kit (Agilent) with enrichment using SureSelect XT Clinical Research Exome v.2 probes (Agilent) on an Illumina HiSeq 4000 (Illumina). A 380‐gene panel associated with cardiovascular disease was analyzed on the Health in Code platform.
10
Genotyping of Adamtsl2 Mutation in Mice
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Genomic DNA extracted from the liver of stb/stb and +/+ mice were used to prepare a paired-end library using SureSelectXT Library Prep Kit (Agilent, Japan). Sequencing was performed on Illumina Novaseq 6000 with 150 bp paired-end reads. CLC Genomics Workbench software 21.0.5 was used for trimming, mapping to the mouse reference genome and identifying variants such as the single nucleotide variants, insertions/deletions and splice-site variants.
To detect the mutation in the genomic DNA of mice, PCR fragments, including exon 15 of Adamtsl2, were amplified from genomic DNA and analyzed by Sanger sequencing or by restriction enzyme digestion. PCR was performed using mismatch primer (5′-CTGGGGTGGTAGCCTGTTCCTGAG-3′ and 5′-ACCGGTCCCCAGTCCGAGAGAGT-3′) under the following conditions: 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 65 °C for 20 s, and 72 °C for 25 s. Amplified fragments were reacted with HinfI and the fragment size was checked by 3.5% agarose gel electrophoresis.
To detect the mutation in the genomic DNA of mice, PCR fragments, including exon 15 of Adamtsl2, were amplified from genomic DNA and analyzed by Sanger sequencing or by restriction enzyme digestion. PCR was performed using mismatch primer (5′-CTGGGGTGGTAGCCTGTTCCTGAG-3′ and 5′-ACCGGTCCCCAGTCCGAGAGAGT-3′) under the following conditions: 94 °C for 2 min followed by 35 cycles of 94 °C for 30 s, 65 °C for 20 s, and 72 °C for 25 s. Amplified fragments were reacted with HinfI and the fragment size was checked by 3.5% agarose gel electrophoresis.
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