High-depth Targeted Sequencing for CRC

Targeted high-depth sequencing was performed for genomic areas comprising 100 genes using a custom-designed CRC panel (Table S1) [16 (link)]. From each sample, 10 ng of DNA were used for multiplexed PCR amplification with the Ion Ampliseq Library kit (ThermoFisher Scientific), using two amplicon pools per DNA sample. Samples were ligated to Ion Xpress Barcode Adapters (ThermoFisher Scientific) and purified using Agencourt AMPure beads (Beckman Coulter). All six samples were combined on a 316v2 chip and sequenced on an Ion Torrent PGM device (ThermoFisher) with an average read depth of 580–700.

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Pfohl U., Loskutov J., Bashir S., Kühn R., Herter P., Templin M., Mamlouk S., Belanov S., Linnebacher M., Bürtin F., Vetter M., Reinhard C., Wedeken L, & Regenbrecht C.R. (2022). A RAS-Independent Biomarker Panel to Reliably Predict Response to MEK Inhibition in Colorectal Cancer. Cancers, 14(13), 3252.

Publication 2022

Ion Ampliseq Library kit Ion Xpress Barcode Adapters Agencourt AMPure beads

Chip Device Genes Genomic Library Multiplexed pcr

Corresponding Organization : University of Tübingen

Other organizations : Goethe University Frankfurt, Max Delbrück Center, Charité - Universitätsmedizin Berlin, University of Helsinki, University of Rostock, University Hospital of Basel, Kantonsspital Baselland

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Variable analysis

independent variables

Targeted high-depth sequencing of genomic areas comprising 100 genes using a custom-designed CRC panel

dependent variables

Average read depth of 580–700

control variables

10 ng of DNA used for multiplexed PCR amplification
Ion Ampliseq Library kit (ThermoFisher Scientific) used for amplification
Two amplicon pools per DNA sample
Samples ligated to Ion Xpress Barcode Adapters (ThermoFisher Scientific)
Samples purified using Agencourt AMPure beads (Beckman Coulter)
All six samples combined on a 316v2 chip
Sequenced on an Ion Torrent PGM device (ThermoFisher)

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