DNA concentration and purity was determined using Qubit (Thermo Fisher Scientific) and Agilent 2100 bioanalzyer analysis. Genomic DNA (200 ng) was sheared using Covaris S220 at 150bp. Sheared DNA was used to construct the exome library following Agilent SureSelect XT Target Enrichment System for Illumina Paired End Multiplexed Sequencing Library (cat# G9641B). Sheared DNA was end repaired followed by addition of adapter tags to construct DNA libraries through PCR amplification. Exome capture was done through hybridization using XT5 probe. Resulting captured libraries were indexed and purified. The cDNA library was validated on the Agilent 2100 bioanalzyer using DNA-1000 chip. Libraries were sequenced on the Illumina HiSeq 2000 with 125 bp paired-end reads. We obtained an average of 400X and 200X sequencing coverage for the cancer and normal exomes, respectively.
Sureselectxt target enrichment system for paired end multiplexed sequencing library
Manufactured by Illumina
Sourced in United States
The SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library is a laboratory equipment product designed for targeted DNA sequencing. It allows for the selective capture and enrichment of specific genomic regions of interest prior to sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Agilent 2100 bioanalzyer, by Agilent Technologies (2 mentions)
Hiseq instrument, by Illumina (2 mentions)
Human gdna, by Promega (2 mentions)
Le220 plus, by Covaris (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Hiseq 3000 4000 sbs kit, by Illumina (1 mentions)
Hiseq 3000 4000 pe cluster kit, by Illumina (1 mentions)
Hiseq 3000 instrument, by Illumina (1 mentions)
Truseq dna pcr free lt library prep kit, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Sureselect all exon v6 kit, by Agilent Technologies (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qiaamp dna ffpe kit, by Qiagen (1 mentions)
14 protocols using sureselectxt target enrichment system for paired end multiplexed sequencing library
1
Exome Sequencing of Cancer and Normal Samples
2
Whole Exome Sequencing of Adrenocortical Carcinoma
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Fresh frozen samples were checked and selected with H&E stainings for high tumor cellularity. For DNA from fresh-frozen samples, library preparation was performed according to Agilent’s “SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library” kit, whereas for DNA from FFPE tissue the “SureSelect Automated Library Prep and Capture System SureSelectXT Automated Target Enrichment for Illumina Paired-End Multiplexed Sequencing” kit was used. Samples were run paired-end (125 bp) on a HiSeq 2000 v4. Alignment of data were processed by the following parameters: reference genome: hs37d5, alignment program: bwa-0.7.8 mem, alignment parameter: -T 0, duplication marking program: picard-1.125, default duplication marking program parameters were used (https://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicates ). Alterations that are likely to be benign, so called FLAGS66 (link) were excluded. Previously published WES studies of ACC13 (link), 14 (link) were utilized as a comparison to the here generated WES results. MutSigCV was used to calculate significantly recurrent mutations67 (link).
3
Chromosome 15 Interactions Profiling
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Cells (20 × 106) were cross-linked with 1% formaldehyde (EMS, 15710) for 10 min at room temperature and quenched with glycine (final concentration, 0.125 M). Cells were lysed in 1 M Tris-HCl pH 8.0, 5 M NaCl and 10% NP40 and complete protease inhibitor (Sigma-Aldrich, 11836170001) and enzymatically digested using 1,000 U of MboI (NEB, R0147). Digested chromatin was then ligated at 16 °C with 10,000 U of T4 DNA ligase (NEB, M0202) in ligase buffer supplemented with 10% Triton X-100 (Sigma-Aldrich, T8787) and 240 µg of BSA (NEB, B9000). Ligated samples were de-cross-linked with 400 μg proteinase K (Macherey Nagel, 740506) at 65 °C and phenol–chloroform purified. 3C library preparation and target enrichment using a custom-designed collection of 6,979 biotinylated RNA ‘baits’ targeting single MboI restriction fragments chromosome 15: 10283500–13195800 (mm9) (Supplementary Table 2 ; Agilent Technologies; designed as in ref. 57 (link)) were performed according to the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library protocol. The only exceptions were the use of 9 µg of 3C input material (instead of 3 µg) and shearing of DNA using Covaris sonication with the following settings: duty factor: 10%; peak incident power: 175; cycles per burst: 200; treatment time: 480 s; bath temperature: 4 °C to 8 °C).
4
Exome Sequencing for Genetic Diagnosis
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The selection of patients to be subjected to CES was based on three main criteria: (i) the lack of genetic diagnosis by PS; (ii) the availability of DNA from at least three relatives; and (iii) the quality of DNA samples. Thus, eleven index patients were selected (Figure 1 ) and captured using the SureSelectXT Focused Exome design (Agilent Technologies, Santa Clara, CA, USA), consisting of exonic regions of ~4800 genes (12 Mb) that have been associated with diseases in databases as HGMD, OMIM and ClinVar. Libraries were prepared following the “SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library” protocol (Version B4, Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed in a Hiseq3000 instrument (Illumina, San Diego, CA, USA) using a HiSeq 3000/4000 SBS Kit (300 cycles) and a HiSeq 3000/4000 PE Cluster Kit.
5
Whole Exome Sequencing of Melanoma
6
Metagenome and Resistome Library Preparation
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DNA was extracted from samples using the PowerMax Soil DNA Isolation Kit according to manufacturer’s instructions, with minor modifications (see Additional file 1 : Supplementary Methods). Library preparation of Metagenome and Resistome libraries followed standard commercial kit protocols, with some modifications. For the Metagenome libraries, the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina) was used, while resistome libraries were created using the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent Technologies) with the custom-designed MEGaRICH bait set. The Resistome-UMI and Metagenome-UMI libraries were created using a protocol that incorporated dual-indexed UMI adapters (see Additional file 13 ) into sequence libraries [23 (link)], with modifications to allow for integration with Agilent SureSelect protocols (see Additional file 1 : Supplementary Methods for details).
7
RNA Bait Capture of Chlamydia trachomatis
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We used a methodology for RNA bait capture of C. trachomatis described in detail by Bowden et al. (2021) (link). Human gDNA (Promega, San Luis Obispo, CA) was added to the extracted gDNA from the clinical swabs to reach a total input of 3 μg/130 μL for fragmentation and library prep. Samples were sheared on the Covaris LE220 plus (Covaris, Woburn, MA). After shearing and magnetic bead purification, the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (VC2 Dec 2018) and all recommended quality control steps were performed on all gDNA samples. The 2.698 Mbp RNA bait library consisted of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes (Bowden et al., 2021 (link))(Agilent Technologies, INC, Santa Clara, CA, reference: ELID: 3173001). A 16-hour incubation at 65°C was performed for RNA bait library hybridization. Post-capture PCR cycling was set at 12 cycles based on a capture library size > 1.5 Mb. The libraries were paired end sequenced for 150 nt using an Illumina HiSeq instrument. Sequence data from this project was submitted to the NCBI Sequence Read Archive under the BioProject accession ID: PRJNA609714 .
8
RNA Bait Capture of Chlamydia trachomatis
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We used a methodology for RNA bait capture of C. trachomatis described in detail by Bowden et al(19 ). Human gDNA (Promega, San Luis Obispo, CA) was added to the extracted gDNA from the clinical swabs to reach a total input of 3 μg/130uL for fragmentation and library prep. Samples were sheared on the Covaris LE220 plus (Covaris, Woburn, MA). After shearing and magnetic bead purification, the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (VC2 Dec 2018) and all recommended quality control steps were performed on all gDNA samples. The 2.698 Mbp RNA bait library consisted of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes(19 )(Agilent Technologies, INC, Santa Clara, CA, reference: ELID: 3173001). A 16-hour incubation at 65°C was performed for RNA bait library hybridization. Post-capture PCR cycling was set at 12 cycles based on a capture library size > 1.5 Mb. The libraries were paired end sequenced for 150 nt using an Illumina HiSeq instrument. Sequence data from this project was submitted to the NCBI Sequence Read Archive under the BioProject accession ID: PRJNA609714
9
Capture Hi-C for Profiling Arabidopsis Genome Architecture
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For Capture Hi-C, in situ Hi-C libraries from shoot and root of Col-0, ref6, and clf were first generated as in Concia et al. (2020) (link). The quality and the DNA concentration of the libraries were assessed with an Agilent 2100 Bioanalyzer (Agilent). Capture was then performed using SureSelect XT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent) according to the manufacturer's recommendation. To this end, 52,911 custom probes were designed, covering 2.658 Mbp and 4650 genes (Supplemental Table S25 ). Capture was performed using 1 µg of in situ Hi-C libraries and following the manufacturer's recommendation. Quality control and sequencing of the libraries were performed in the same way as for HiChIP assays.
10
Exome Sequencing Using SureSelect
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The library prep was done with the SureSelect XT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent Technologies, Santa Clara, CA, USA). Exonic regions were captured using SureSelect All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. Exonic regions, covering 60 Mb of protein-coding bases, were captured through hybrid captures. Sequencing was performed on an Illumina HiSeq 2500 by 125-base paired-end at Novogen, Sacramento, California.
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