CLIA-certified WES was conducted by the Clinical Research Sequencing Platform, Broad Institute; CLIA #:22D2055652. Library construction from surgical melanoma specimens and matched germline DNA of all 10 patients was performed as previously described25 (link). Genomic DNA was sheared, end repaired, ligated with barcoded Illumina sequencing adapters, amplified, and size selected. For Patients 1, 2, 3, 4, 7 and 9, whole exome capture was performed using the Agilent SureSelect Human All Exon 44Mb v2.0 bait set (Agilent Technologies)26 (link). For Patients 5, 6, 8, and 10, WES was performed using the Illumina Nextera Rapid Capture Exome v1.2 bait set. The Illumina exome specifically targets approximately 37.7Mb of mainly exonic regions made up of all targets from the Agilent exome design (Agilent SureSelect All Exon V2), all coding regions of Gencode V11 genes, and all coding regions of RefSeq gene and KnownGene tracks from the UCSC genome browser (http://genome.ucsc.edu ). Resulting libraries were then qPCR quantified, pooled, and sequenced with 76 base paired-end reads using HiSeq 2000 or 2500 sequencers (Illumina). Pooled libraries were normalized to 2nM and denatured using 0.2 N NaOH prior to sequencing. Data were analyzed using the Broad Picard Pipeline which includes de-multiplexing, duplicate marking, and data aggregation.
Hiseq 2000 or 2500 sequencers
Manufactured by Illumina
Sourced in United States
The HiSeq 2000 and HiSeq 2500 are high-throughput DNA sequencing instruments developed by Illumina. They are designed to rapidly sequence large amounts of DNA samples, generating millions of sequence reads per run. The core function of these sequencers is to perform massively parallel sequencing using reversible terminator chemistry, allowing for the determination of nucleotide sequences in DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
Agilent sure select human all exon 44 mb version 2.0 bait set, by Agilent Technologies (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Rneasy mini kit, by Illumina (2 mentions)
Sequencing adapter, by Illumina (1 mentions)
Truseq sbs kit v3 hs, by Illumina (1 mentions)
Hiseq 2500 sequencing platform, by Illumina (1 mentions)
4 protocols using hiseq 2000 or 2500 sequencers
1
Whole Exome Sequencing of Melanoma Specimens
2
Extraction and Sequencing of Tumor RNA and DNA
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For RNA sequencing (RNA-seq), an RNeasy mini kit (Illumina, San Diego, CA, USA) was used to extract the RNA from fresh frozen tumor samples. RNA-seq libraries were constructed using the TruSeq Stranded mRNA Library Prep kit (Illumina) (for cell suspensions). Flow cytometry amplification and sequencing were performed using HiSeq 2500 according to the manufacturer’s instructions.
For whole-exome sequencing (WES), DNA was extracted from fresh-frozen tumor samples or cultured tumor cells using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Genomic DNA was fragmented, end-repaired, and simultaneously ligated to the bar-coded sequencing adapters (Illumina), amplified, and size-selected. Whole-exome capture was performed using Agilent SureSelect Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies, Santa Clara, CA, USA).14 (link) The resulting libraries were quantified using a quantitative polymerase chain reaction (qPCR), and 76 base-paired end reads were generated and sequenced using HiSeq 2000 or 2500 sequencers (Illumina).
For whole-exome sequencing (WES), DNA was extracted from fresh-frozen tumor samples or cultured tumor cells using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). Genomic DNA was fragmented, end-repaired, and simultaneously ligated to the bar-coded sequencing adapters (Illumina), amplified, and size-selected. Whole-exome capture was performed using Agilent SureSelect Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies, Santa Clara, CA, USA).14 (link) The resulting libraries were quantified using a quantitative polymerase chain reaction (qPCR), and 76 base-paired end reads were generated and sequenced using HiSeq 2000 or 2500 sequencers (Illumina).
3
Whole Exome and Transcriptome Sequencing of Tumor Samples
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For WES sequencing, DNA from fresh frozen tumor samples or cells was extracted using the DNeasy Blood and Tissue Kit, which was purchased from Qiagen. The genomic DNA was sheared, end-repaired, ligated to barcoded Illumina sequencing adapters, amplified, and size-selected. Whole-exome capture was performed using an Agilent Sure Select Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies) (21 (link)). The resulting libraries were then quantified by qPCR, pooled, and sequenced using 76-basepaired-end reads obtained with HiSeq 2000 or 2500 sequencers (Illumina).
For RNA sequencing, RNA from fresh frozen tumor samples was extracted using the RNeasy Mini Kit. RNA-seq libraries were prepared using an Illumina TruSeq Stranded mRNA Library Prep Kit (for cell suspensions). Flow cell cluster amplification and sequencing were performed according to the manufacturer’s instructions using either a HiSeq2500.
The sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession code SRP (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB8559404/overview ).
For RNA sequencing, RNA from fresh frozen tumor samples was extracted using the RNeasy Mini Kit. RNA-seq libraries were prepared using an Illumina TruSeq Stranded mRNA Library Prep Kit (for cell suspensions). Flow cell cluster amplification and sequencing were performed according to the manufacturer’s instructions using either a HiSeq2500.
The sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession code SRP (
4
Comprehensive RNA-seq and WES Sequencing
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For RNA sequencing (RNA-seq), RNeasy Mini Kit was utilized to extract RNA from fresh frozen tumor samples. The construction of RNAseq libraries were performed using the TruSeq Stranded mRNA Library Prep kit (Illumina) (for cell suspensions). All libraries were sequenced paired-end 50 nt on an Illumina HiSeq 2500 sequencing platform by using two Illumina-based TruSeq SBS Kits v3-HS 50 cycles. Paired ends (100 nt) were sequenced using TruSeq SBS Kit v3-HS (Illumina) (200-cycles) to distribute the MZ-GaBa-018 cell line and its matched PBMC genome-wide sequencing library in 4 lanes.
For WES sequencing, DNA was extracted from the fresh-frozen tumor samples or cultured tumor cells using DNeasy Blood and Tissue Kit, purchased from Qiagen. Genomic DNA was cut, end-repaired, simultaneously connected to the bar-coded Illumina sequencing adapters, ampli ed, and size-selected. Whole-exome capture was performed using Agilent Sure Select Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies), The resulting libraries were quanti ed by qPCR, and 76-base paired-end reads were pooled and sequenced using HiSeq 2000 or 2500 sequencers (Illumina).
For WES sequencing, DNA was extracted from the fresh-frozen tumor samples or cultured tumor cells using DNeasy Blood and Tissue Kit, purchased from Qiagen. Genomic DNA was cut, end-repaired, simultaneously connected to the bar-coded Illumina sequencing adapters, ampli ed, and size-selected. Whole-exome capture was performed using Agilent Sure Select Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies), The resulting libraries were quanti ed by qPCR, and 76-base paired-end reads were pooled and sequenced using HiSeq 2000 or 2500 sequencers (Illumina).
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