Ion xpress barcode adaptors

To ensure mitochondrial genome specificity and thereby avoiding amplification of nuclear copies of mitochondrial-derived DNA (NuMTs), we performed a dual-amplicon long-range pre-amplification of the mitochondrial genome prior to sequencing on the Ion Torrent PGM instrument, as previously described [22 (link)]. In brief, after quantification, the 7.2 kilobase (kb) and 9.7 kb amplified products were combined in a 7:13 ratio, 200 bp libraries prepared using the Ion Xpress Plus Fragment Kit and Ion Xpress™ Barcode Adaptors (ThermoFisher), and six to eight matched normal-tumor pairs pooled and run on a 318v2 Chip. We generated per patient an average of 80 Megabytes (MB) of data, with an average coverage depth of more than 2000 X per mitochondrial genome.

Prior to HTS, tdc amplicon libraries were prepared using the Ion Xpress Plus Fragment Library Kit (Life Technologies, Dublin, Ireland). The hdc libraries, for which fragmentation was not required, were prepared using the Ion Plus Fragment Library Kit (Life Technologies, Dublin, Ireland). Libraries were then barcoded, prior to sequencing, using the Ion Xpress Barcode Adaptors (Life Technologies, Dublin, Ireland). Amplicons libraries were assessed for size distribution and concentration using a Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA USA). Following library quantification and equimolar pooling, the Ion OneTouch 2 system was used to prepare template positive Ion Sphere Particles (ISP) containing the clonally amplified DNA libraries using the Ion PGM Template OT2 400 kit which allows for < 400 bp reads. Enrichment of the template positive ISP’s was performed using the Ion OneTouch ES. An enrichment percentage of 18 % was obtained. Sequencing was performed on the Ion Torrent PGM (Life Technologies, Dublin, Ireland) using an Ion 318v2 chip and the Ion PGM Sequencing 400 kit (Life Technologies, Dublin, Ireland) at the Teagasc Next Generation Sequencing suite as per the manufacturer’s guidelines.

The NGS assay for extended RAS and BRAF mutations was designed to detect mutations in codons 12, 13, 59, 61, 117, and 146 of KRAS and NRAS and in codon 600 of BRAF using an Ion Torrent PGM semiconductor sequencer (Life Technologies). The PCR primers were designed using Assay Designer 3.1 (Sequenom, San Diego, CA). Multiplex PCR was performed in a volume of 20 μL containing 10 ng of DNA, 4 U of Taq polymerase (Sequenom), 500 μM of each deoxynucleoside triphosphate (Sequenom), and 0.2 μM of each PCR primer. Thermal cycling was performed at 95°C for 15 min, followed by 45 cycles of 94°C for 20 sec, 56°C for 30 sec, and 72°C for 60 sec, followed by a final extension of 72°C for 3 min. The PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA), followed by the preparation of a barcoded DNA library using the Ion plus Fragment Library Kit (Life Technologies) and IonXpress Barcode Adaptors (Life Technologies). The quantified DNA libraries were pooled and sequenced using the Ion PGM 200 Sequencing Kit v2 and the Ion 318 v2 Chip Kit. The DNA sequencing data were accessed through the Torrent Suite v.4.0 software program. The reads were aligned against the hg19 human reference genome, and the variants were called using the plug-in Variant Caller (ver.4.0, Life Technologies).