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Agilent sureselect target enrichment system

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The Agilent SureSelect target enrichment system is a versatile platform designed to selectively capture and sequence specific genomic regions of interest. It enables efficient and cost-effective targeted sequencing by isolating and enriching user-defined DNA or RNA sequences from complex samples.

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Lab products found in correlation

Qiaamp dna blood mini kit, by Qiagen (4 mentions) Neb phusion hf master mix, by New England Biolabs (2 mentions) Acoustic solubilizer, by Covaris (2 mentions) Ingenuity variant analysis, by Qiagen (2 mentions) Hiseq 2500 system, by Illumina (2 mentions) Xt inherited disease panel cat no 5190 7519, by Agilent Technologies (2 mentions) Hiseq 2500, by Illumina (1 mentions) Gentra puregene kit, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Miseq sequencer, by Illumina (1 mentions) Sureselectxt reagent kit, by Agilent Technologies (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions) Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Hiseq system, by Illumina (1 mentions) Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions) Hiseq 2000 system, by Illumina (1 mentions) Cbot system, by Illumina (1 mentions) Sequence control software scs, by Illumina (1 mentions) Nextgene, by SoftGenetics (1 mentions) Hiseq 2000, by Illumina (1 mentions)

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SureSelect Target Enrichment System (74 citations) Agilent SureSelect system (5 citations) Agilent Mouse SureSelect Methyl-seq Target Enrichment System (4 citations) Agilent SureSelect Target Enrichment System Kit (2 citations) Agilent SureSelect Target Enrichment System (Human All Exon V6 Kit). (2 citations)

9 protocols using agilent sureselect target enrichment system

1

Targeted Genomic Sequencing of PKD1 Gene

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After preparing libraries from patient-derived gDNA, library preparation, targeted genomic enrichment and massively parallel sequencing (MPS) were completed as we have described [14 (link)]. In brief, libraries were prepared using a modification of the solution-based Agilent SureSelect target enrichment system (Agilent Technologies, Santa Clara, CA, USA) with liquid-handling automation. Hybridization and capture with RNA baits were followed by a second amplification. Before pooling for sequencing, all samples were bar coded and multiplexed. Sequencing was done using Illumina HiSeq (pool of 48 samples) or MiSeq (pools of five samples) instrumentation (Illumina Inc., San Diego, CA, USA). Sanger sequencing was used to amplify and resolve exons 1–34 of PKD1 [15 (link), 16 (link)].
Mansilla M.A., Sompallae R.R., Nishimura C.J., Kwitek A.E., Kimble M.J., Freese M.E., Campbell C.A., Smith R.J, & Thomas C.P. (2019). Targeted broad-based genetic testing by next-generation sequencing informs diagnosis and facilitates management in patients with kidney diseases. Nephrology Dialysis Transplantation, 36(2), 295-305.
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2

Genomic DNA Extraction and Complement Gene Analysis

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For each sample, genomic DNA was extracted from peripheral blood using the Gentra Puregene Kit (Qiagen Inc., Valencia, CA); integrity was evaluated by 1% agarose gel electrophoresis. To ensure DNA samples met our minimal quality metrics of 1.8 for 260/280 and 260/230 ratios, absorbance was measured at 230:260:280 using a NanoDrop 1000 spectrophotometer (Thermo Scientific Inc., Wilmington, DE). DNA concentration was determined using the Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA). Complement genes were analyzed by capturing the coding sequence and flanking splice sites using the Agilent SureSelect Target Enrichment System (Agilent Technologies Inc., Santa Clara, CA), as we have described.15 (link) Library preparation was performed with SureSelect TGE baits and SureSelectXT Reagent Kits (Agilent Technologies Inc., Santa Clara, CA) following the manufacturer’s protocol. Preparation was automated using a Zephyr Workstation (PerkinElmer, Waltham, MI). Library quality and concentration were evaluated using a Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA). Libraries passing quality control step were pooled and sequenced on a HiSeq 2500 (Illumina Inc., San Diego, CA) or a MiSeq Sequencer (Illumina Inc., San Diego, CA).
Ravindran A., Fervenza F.C., Smith R.J., De Vriese A.S, & Sethi S. (2018). C3 Glomerulopathy: 10-Years Experience at the Mayo Clinic. Mayo Clinic proceedings, 93(8), 991-1008.
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3

Targeted Genomic Enrichment for NSHL

Cited 2 times
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The TGE of all exons of all genes implicated in NSHL, including NSHL mimics, was completed as described, targeting 66 or 89 genes as part of the OtoSCOPE® v4 or v5 platforms, respectively (see Supporting Information, Table S1 for full list)(5 (link)). Libraries were prepared using a modification of the solution-based Agilent SureSelect target enrichment system (Agilent Technologies, Santa Clara, CA)(6 (link)). In brief, 3μg of gDNA was randomly fragmented to an average size of 250 bp (Covaris Acoustic Solubilizer; Covaris Inc., Woburn, MA), and adaptor ligated before the first amplification. Hybridization and capture with RNA baits was followed by a second amplification before pooling for sequencing. Minimal amplification was used – typically 8 cycles for the pre-hybridization PCR (range 8–10 cycles) using NEB Phusion HF Master Mix (New England BioLabs Inc, Ipswich, MA) and 14 cycles for the post-hybridization PCR (range 12–16 cycles) using Agilent Herculase II Fusion DNA Polymerase. All samples were barcoded and multiplexed before sequencing on either an Illumina MiSeq or HiSeq system (Illumina Inc, San Diego, CA) in pools of 4–6 or 48, respectively, using 100-bp paired-end reads.
Moteki H., Azaiez H., Booth K.T., Shearer A.E., Sloan C.M., Kolbe D.L., Nishio S.Y., Hattori M., Usami S.I, & Smith R.J. (2015). Comprehensive genetic testing with ethnic-specific filtering by allele frequency in a Japanese hearing-loss population. Clinical genetics, 89(4), 466-472.
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4

Whole Exome Sequencing of Genomic DNA

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The genomic DNA of the patient and parents was isolated from peripheral blood samples using a QIAamp DNA Blood Mini kit® (Qiagen GmbH, Hilden, Germany). A total of 3 µg DNA from the patient was sheared to produce 150–200 base pair (bp) fragments. An adapter-ligated library was prepared using an Agilent SureSelect Target Enrichment system (Agilent Technologies, Inc., Santa Clara, CA, USA) and enrichment of coding exons and flanking intronic regions was performed using Agilent SureSelect Human All Exon V6 reagent (Agilent Technologies, Inc.), according to the manufacturer's protocol. Clusters were subsequently generated using isothermal bridge amplification with an Illumina cBot system (Illumina Inc., San Diego, CA, USA) and sequencing was performed using an Illumina HiSeq 2000 system (Illumina, Inc.).
Base calling and assessment of sequence read quality was performed using Illumina Sequence Control Software (SCS; Illumina, Inc.) with real-time analysis. Alignment of sequence reads to a reference human genome (Human 37.3, single nucleotide polymorphism 135) was performed using NextGENe® (SoftGenetics, LLC., State College, PA, USA). All single-nucleotide variants (SNVs) and indels were saved in a variant call format and uploaded for Ingenuity® Variant Analysis™ (Ingenuity Systems; Qiagen GmbH) for biological analysis and interpretation.
Ge Y., Li N., Wang Z., Wang J, & Cai H. (2017). Novel variant in the FGD1 gene causing Aarskog-Scott syndrome. Experimental and Therapeutic Medicine, 13(6), 2623-2628.
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5

Inherited Disease Genomic DNA Sequencing Protocol

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Patients’ genomic DNA was isolated from 2‐ml peripheral blood samples using a QIAamp Blood DNA Mini Kit® (Qiagen GmbH, Hilden, Germany). Three micrograms of DNA was processed through shearing using a Covarias® M220 Ultrasonicator system (Covaris, Inc. Woburn, MA, USA) to pieces of 150–200 bp in size. An adapter‐ligated library was produced with Agilent SureSelect Target Enrichment System (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer's instructions. The capture library was performed using an XT Inherited Disease Panel (cat No. 5190–7519, Agilent technologies, Inc.), containing 2,742 genes known to cause inherited disorders, covering only 10.5 Mb. Clusters were then generated by isothermal bridge amplification using an Illumina cBot station, and sequencing was performed on an Illumina HiSeq 2500 System (Illumina, Inc., San Diego, CA, USA). The raw data (fastq file) for each patient were obtained for CNV identification. The average sequencing depth of data used was 122, and more that 95.6% of targeted region was covered with 20 reads.
Yao R., Yu T., Qing Y., Wang J, & Shen Y. (2018). Evaluation of copy number variant detection from panel‐based next‐generation sequencing data. Molecular Genetics & Genomic Medicine, 7(1), e00513.
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6

Whole Exome Sequencing of Patients

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Peripheral blood samples were collected from the patients and their parents after informed consent was obtained. WES was performed on these patients as mentioned before [12 (link), 13 (link)]. A QIAamp DNA Blood Mini kit® (Qiagen GmbH, Hilden, Germany) was used to isolate genomic DNA. Library was established with an Agilent SureSelect Target Enrichment system (Agilent Technologies, Inc., Santa Clara, CA, USA). And the system of Illumina HiSeq 2000 (Illumina, Inc.) and an Illumina cBot (Illumina Inc., San Diego, CA, USA) were used to sequence and generate clusters. All variants detected were filtered and annotated by Ingenuity Variant Analysis (Ingenuity Systems, Redwood City, CA, USA). Finally, Sanger sequencing was used to confirm the variants detected by WES comparing to the individuals’ parents. The potential pathogenicity of the missense variant was evaluated using three in silico prediction methods: SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and MutationTaster (http://www.mutationtaster.org/ChrPos.html).
Zhang Q., Chang G., Tang Y., Gu S., Ding Y., Chen Y., Wang Y., Liu S., Wang J, & Wang X. (2023). Genotypic and phenotypic features of dyslipidemia in a sample of pediatric patients in China. BMC Pediatrics, 23, 138.
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7

Targeted Gene Enrichment for Non-syndromic SNHL

Cited 1 time
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TGE of all exons of all genes implicated in non-syndromic SNHL, including non-syndromic SNHL mimics, was completed as described, targeting 89 genes as part of the OtoSCOPE® v5 platform. Libraries were prepared using a modification of the solution-based Agilent SureSelect target enrichment system (Agilent Technologies, Santa Clara, CA).11 (link)In brief, 3μg gDNA was randomly fragmented to an average size of 250 bp (Covaris Acoustic Solubilizer; Covaris Inc., Woburn, MA), fragment ends were repaired, A-tails were added, and sequencing adaptors were ligated before the first amplification. Solid phase reverse immobilization purifications were performed between each enzymatic reaction. Hybridization and capture with RNA baits was followed by a second amplification before pooling for sequencing. Minimal amplification was used – typically 8 cycles for the prehybridization PCR (range 8–10 cycles) using NEB Phusion HF Master Mix (New England BioLabs Inc, Ipswich, MA) and 14 cycles for the post-hybridization PCR (range 12–16 cycles) using Agilent Herculase II Fusion DNA Polymerase. All samples were barcoded and multiplexed before sequencing on either an Illumina MiSeq or HiSeq (Illumina Inc, San Diego, CA) in pools of 4-6 or 48, respectively, using 100-bp paired-end reads.
Sakuma N., Moteki H., Azaiez H., Booth K.T., Takahashi M., Arai Y., Shearer A.E., Sloan C.M., Nishio S.Y., Kolbe D.L., Iwasaki S., Oridate N., Smith R.J, & Usami S.I. (2015). Novel PTPRQ mutations identified in three congenital hearing loss patients with various types of hearing loss. The Annals of otology, rhinology, and laryngology, 124(1 0), 184S-192S.
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8

Genomic DNA Extraction and Sequencing for Inherited Disease

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The patient's and her parents' genomic DNA were extracted from 2-ml peripheral blood samples using the QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The DNA concentration and purity were measured using an Invitrogen Qubit dsDNA detection kit and a Qubit4 fluorometer (Carlsbad, CA, USA). The Agilent Sure Select Target Enrichment System (Agilent Technologies Inc., Santa Clara, CA, US) was used to produce an adapter-ligated library according to the manufacturer's instructions. An XT Inherited Disease Panel (cat No. 5190–7519, Agilent Technologies Inc.) containing 2,742 genes was used to create the capture library. The clusters were then produced using an Illumina cBot Station and sequenced on an Illumina HiSeq 2,500 System (Illumina Inc., San Diego, CA, US). Using human genome hg19 as the reference, alignment of sequence and repeated labeling were performed using BWM version 0.7.17 (http://bio-bwa.sourceforge.net/) and Picard bioinformatics software version 2.5.0 (https://broadinstitute.github.io/picard/) for biological analysis and interpretation. GATK 4.0.0.0 (https://gatk.broadinstitute.org/hc/en-us/sections/360007407851-4-0-0-0) and Samtools 1.8 (https://sourceforge.net/projects/samtools/files/samtools/1.8/) were used to identify mutation sites.
Ni Y., Chen X., Sun Y., Pan J., Tang C, & Yuan T. (2022). Modulation of PC1/3 activity by a rare double-site homozygous mutation. Frontiers in Pediatrics, 10, 1026707.
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9

Next-Gen Sequencing Exome Capture Protocol

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Next generation sequencing libraries were built according to established laboratory protocols at the Science for Life Laboratory Stockholm and the Institute for Molecular Medicine Finland (FIMM). The exome libraries were processed according to the Agilent SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA) for Illumina Paired-End Sequencing Libraries (Illumina, San Diego, CA, USA) using the SureSelect Human All Exon V5 capture library (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced with 101 bp read length (HiSeq1500 sequencing platform, Illumina, San Diego, CA, USA). The read mapping, variant calling and genome annotation were performed as described previously (28 (link)). We focused the search on the haplotype that segregated with the phenotype, prioritizing rare, heterozygous coding variants that negatively affect conserved residues and shared by both whole genome sequenced individuals. Only one such variant was identified, localized within the TMEM173 gene. The G207E variant was absent from all major public [The Exome Aggregation Consortium (ExAC), 1,000 Genomes, NHLBI Exome variant server and UK TWIN and ALSPAC study cohorts (2–4)], as well as in-house databases.
Keskitalo S., Haapaniemi E., Einarsdottir E., Rajamäki K., Heikkilä H., Ilander M., Pöyhönen M., Morgunova E., Hokynar K., Lagström S., Kivirikko S., Mustjoki S., Eklund K., Saarela J., Kere J., Seppänen M.R., Ranki A., Hannula-Jouppi K, & Varjosalo M. (2019). Novel TMEM173 Mutation and the Role of Disease Modifying Alleles. Frontiers in Immunology, 10, 2770.
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