Onetouch 2

Libraries of 16S DNA were constructed based on PCR amplification of 7 of the 9 hypervariable regions of the 16S rDNA gene (V2, V3, V4, V6–V9). Amplification was performed in two independent reactions using the 16S Metagenomics™ system according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) in a Verity™ thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). An equimolar mixture using the amplification products was prepared, and 50 nanograms were used to construct the 16S rDNA libraries with the Ion Plus Fragment Library commercial system and the Ion Xpress barcode adapters (Thermo Fisher Scientific). Library purification was carried out using the Agentcourt AMPure XP system according to the manufacturer’s instructions (Beckman Coulter, Brea, CA, USA) and quantified with a highly sensitive DNA commercial system and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Library concentration was adjusted to 26 pM followed by PCR amplification of the PCR emulsion using a volume of 25 µL of the equimolar mixture for all samples (One-Touch 2, Thermo Fisher Scientific, Waltham, MA, USA) and enriched with the OneTouch Enrichment system (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was carried out using the Ion S5™ system (Thermo Fisher Scientific, Waltham, MA, USA).

Ten nanograms of DNA was used for multiplex PCR amplification. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quality of the obtained libraries was evaluated using Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, Palo Alto, CA, USA). Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific), loaded with a 316 or 318v2 chip as per the manufacturer’s protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was completed using Torrent Suite Software v3.6 (Thermo Fisher Scientific). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific).

Ten nanogram of DNA were used for multiplex PCR amplification. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The quality of obtained library was evaluated by the Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, USA).
Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific, USA). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, USA), loading with 316™ or 318™v2 chip as per manufacturer's protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was done using the Torrent Suite Software v.3.6 (Thermo Fisher Scientific, USA). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific, USA). Alignments were visually verified with the Integrative Genomics Viewer; v.2.3. The mean coverage achieved was 1361-fold and 2338-fold in the tumor tissues for GDF15 and TP53 sequencing, respectively, and the same deep sequencing was done (two sample in 316™ chip or four sample in 318™v2 chip) on the control tissues. 90% and 95% of the targeted bases were represented by at least 10 reads for GDF15 and TP53, respectively.