Human genomic DNA was extracted from tissue samples using DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. The genomic DNA was then randomly broken to a length of 180-280bp fragments by Covaris. Next, Sure Select Human All Exon V5/V6 kit (Agilent, Santa Clara, USA) was used for library construction and capture experiments according to the manufacturer’s instructions. A total of 334378 exons for 20965 genes were captured by magnetic beads with streptomycin. After DNA quality evaluation, captured DNA libraries underwent sequencing on Illumina HiSeq 4000, which resulted in 150bp paired-end reads from the end of the fragments.
Sure select human all exon v5 v6 kit
Manufactured by Agilent Technologies
Sourced in United States
The SureSelect Human All Exon V5/V6 kit is a targeted enrichment solution designed for capturing and sequencing the protein-coding regions of the human genome, known as the exome. The kit utilizes RNA baits to selectively capture and enrich the exonic regions, enabling efficient and cost-effective analysis of the human exome.
Automatically generated - may contain errors
Lab products found in correlation
Fragmentation apparatus, by Covaris (2 mentions)
Agilent 2100, by Agilent Technologies (2 mentions)
Hiseq 4000, by Illumina (1 mentions)
Dna ffpe tissue kit, by Qiagen (1 mentions)
Hiseq pe150, by Illumina (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Humancytosnp 12 beadchip platform, by Illumina (1 mentions)
5 protocols using sure select human all exon v5 v6 kit
1
Whole-Exome Sequencing from FFPE Samples
2
Exome Sequencing of RCC4 and HK2 Cells
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RCC4 and HK2 cells were grown in 100 mm dishes until 80% confluency. Genomic DNA were extracted with TIANamp Genomic DNA Kit (Tiangen Biotech, DP304-03). Using agarose gel electrophoresis to analyze the integrity and purity of DNA, and the concentration was determined by Qubit 2.0. The exome sequencing were performed by Beijing Novogene Biotech Co., Ltd. In brief, DNA library were constructed using Agilent SureSelect Human All Exon V5/V6 Kit, then the quality was assessed using Q-PCR. The sequencing was performed with Illumina HiSeq PE150. Raw data were processed and aligned to human reference genome (GRCh37.p13) through BWA and Samblaster software. The mutations were analyzed by SAMtools and ANNOVAR software.
3
Exome Sequencing Library Preparation
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Qubit 2.0 is used to accurately quantify the concentration of DNA samples. DNA samples with a DNA concentration of ≥ 20 ng/µL and a total amount of 0.6 µg or more are used to build the library. Genomic DNA was randomly fragmented into 180–280 bp fragments using a Covaris fragmentation apparatus. The Agilent sureselect human all exon V5/v6 kit was used for the construction and capture of genomic DNA library. The library with a specific index was hybridized with biotin labeled probe in the liquid phase. Magnetic beads with streptomycin were used to capture the exons, which were linearly amplified by PCR for library quality inspection. Qubit 2.0, Q-PCR, and Agilent 2100 were used to quantify and detect the library.
4
Identifying Runs of Homozygosity Using Genotyping and Exome Sequencing
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DNA from F3:II-1 and F3:II-2 of family 3 were genotyped with the HumanCytoSNP-12 BeadChip platform (Illumina). The genotypes were evaluated for runs of homozygosity (ROH) >1 Mb via PLINK software14 (link) integrated in in-house software ViVar.15 (link) Resulting ROH were ranked according to their length and number of consecutive homozygous single-nucleotide polymorphisms (SNPs).16 (link) For ES, exome enrichment and sequencing were performed with the Agilent SureSelect Human All exon V5/V6 kit followed by paired-end sequencing on a HiSeq2000 (2 × 100 cycles). The CLC Genomics Workbench version 9.0.1 (CLCBio) was used for read-mapping against the human genome reference (NCBI build37/hg19 version), post-mapping duplicate read removal, coverage analysis, and quality-based variant calling via Alamut (visual version 2.7.2; interactive biosoftware).
5
Targeted Exome Sequencing Library Preparation
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Qubit 2.0 is used to accurately quantify the concentration of DNA samples. DNA samples with a DNA concentration of ≥ 20 ng/ µL and a total amount of 0.6 µg or more are used to build the library. Genomic DNA was randomly fragmented into 180-280 bp fragments using a Covaris fragmentation apparatus. The Agilent sureselect human all exon V5 / v6 kit was used for the construction and capture of genomic DNA library. The library with a speci c index was hybridized with biotin labeled probe in the liquid phase. Magnetic beads with streptomycin were used to capture the exons, which were linearly ampli ed by PCR for library quality inspection. Qubit 2.0, Q-PCR, and Agilent 2100 were used to quantify and detect the library.
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