Using the obtained genomic DNA, we then amplified the target genes by a polymerase chain reaction (PCR) method with Gene Read DNA seq Panel PCR Regent V2 (Qiagen) and Human Comprehensive Cancer Panel (Qiagen). We performed targeted amplicon exome sequencing for 160 cancer-related genes based on the Illumina MiSeq sequencing platform (Illumina, San Diego, USA) with an average depth of coverage of approximately 500 × with at least 80% of bases covered >50 × [[16] (link), [17] (link), [18] (link), [19] (link), [20] (link), [21] (link)]. Sequencing (paired-end, 150 bp) of samples and demultiplexing of libraries was performed by Illumina MiSeq Reporter. The list of 160 genes is shown in Supplementary Table 1 [19 (link),20 (link)]. Details of data analysis are described in Supplementary Methods. Germline variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines [22 (link)], and retained as pathogenic based on the recommendations of ACMG and ClinVar [23 (link)]. Germline secondary findings were disclosed only to those patients who agreed to receive this information.
Gene read dna seq panel pcr regent v2
Manufactured by Qiagen
The Gene Read DNA seq Panel PCR Regent V2 is a laboratory equipment product designed for DNA sequencing workflows. It provides the necessary reagents and materials required for performing polymerase chain reaction (PCR) amplification as part of the DNA sequencing process.
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2 protocols using gene read dna seq panel pcr regent v2
1
Comprehensive Cancer Gene Profiling
2
Targeted Gene Amplification and Sequencing
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Using the obtained genomic DNA, we then amplified the target genes by a polymerase chain reaction (PCR) method with Gene Read DNA seq Panel PCR Regent V2 (Qiagen). To collect the amplified DNA, the PCR products were added to Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA), bound to magnetic beads, and then eluted from the magnetic beads. Terminal repair and adapter ligation were performed using a Gene Read DNA Library Core Kit (Qiagen) to prepare a purified DNA library. The DNA library was amplified by PCR with a GeneRead DNA Amp Kit (Qiagen), and purified using a MinElute PCR Purification Kit (Qiagen). The amplified DNA library was sequenced by Miseq (Illumina).
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