<b>Optimizing an ion semiconductor sequencing data analysis method to identify somatic mutations in the genomes of cancer cells in clinical tissue </b><b>samples </b>

Abstract

Identification of causal genomic alterations is an indispensable step in the implementation of personalized cancer medicine. Analytical methods play a central role in identifying such changes because of the vast amount of data produced by next generation sequencer. Most analytical techniques are designed for the Illumina platform and are therefore suboptimal for analyzing datasets generated by whole exome sequencing (WES) using the Ion Proton System. Accurate identification of somatic mutations requires the characterization of platform-dependent error profiles and genomic properties that affect the accuracy of sequence data as well as platform-oriented optimization of the pipeline. Therefore, we used the Ion Proton System to perform WES of DNAs isolated from tumor and matched control tissues of 1,058 patients with cancer who were treated at the Shizuoka Cancer Center Hospital. Among the initially identified candidate somatic single-nucleotide variants (SNVs), 10,279 were validated by manual inspection of the WES data followed by Sanger sequencing. These validated SNVs were used as an objective standard to determine an optimum cutoff value to improve the pipeline. Using this optimized pipeline analysis, 189,381 SNVs were identified in 1,101 samples. The analytical technique presented here is a useful resource for conducting clinical WES, particularly using semiconductor-based sequencing technology.

Japanese version of The Cancer Genome Atlas, JCGA, established using fresh frozen tumors obtained from 5143 cancer patients

“…WES, CCP and fusion gene panel sequencing were performed using an Ion Proton System (Thermo Fisher Scientific) and transcriptome profiling was performed using an Agilent system (Agilent Technologies). Detailed experimental protocols have been previously described . Sequence data analysis procedures are described in Doc.…”

Germline mismatch repair gene variants analyzed by universal sequencing in Japanese cancer patients

“…Germline DNA was extracted from blood samples obtained soon before the surgery using a QIAamp DNA Blood Kit (QIAGEN, Venlo, Netherlands). WES was performed using an Ion Torrent AmpliSeq Exome RDY Panel kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's recommended protocol . Briefly, 10 ng of DNA was used to prepare the template, and the libraries were prepared automatically using the Ion Chef System (Thermo Fisher Scientific).…”

“…WES was performed using an Ion Torrent AmpliSeq Exome RDY Panel kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's recommended protocol. 30,31 Briefly, 10 ng of DNA was used to prepare the template, and the libraries were prepared automatically using the Ion Chef System (Thermo Fisher Scientific). Libraries were quantified using the quantitative polymerase chain reaction, and 7 pM was sequenced using the Ion Torrent Proton Sequencer (Thermo Fisher Scientific) semiconductor DNA sequencer according to the manufacturer's protocol.…”

<b>Metabolic profiling of gemcitabine- and paclitaxel-treated immortalized human pancreatic cell lines with K-RAS</b>^{<b>G12D </b>}

“…Whole exome sequencing analysis was performed according to previously described methods (23). Briefly, DNA was extracted from 10 6 cells using a kit (Blood & Cell Culture DNA Midi Kit; QIAGEN, Hilden, Germany).…”