Ion s5 sequencer

We performed target deep sequencing of the genomic DNA extracted from the three CRC tissue samples and matched normal samples using a custom NGS panel (OncoChase-AS01, ConnectaGen, Seoul, Korea), targeting 95 cancer-related genes as described elsewhere [10 (link),11 (link)]. Tumor DNA was amplified, digested, and barcoded using the Ion Ampliseq library kit 2.0 (Thermo Fisher Scientific) and Ion Xpress barcode adapter kit (Thermo Fisher Scientific) as described elsewhere [10 (link)]. The libraries were then templated on an Ion Chef system (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef reagents (Thermo Fisher Scientific). The prepared libraries were sequenced on an Ion S5 sequencer using an Ion 530 chip and Ion S5 sequencing reagents (Thermo Fisher Scientific) as described elsewhere [10 (link)].

Library preparation was performed according to manufacturer's instructions. All the purifications were made using AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Library quantification was performed using Ion Library TaqMan^® Quantitation kit (Thermo Fisher) in a StepOnePlus™ qPCR machine (Thermo Fisher). The individual libraries were diluted to a final concentration of 100 pm. The final barcoded libraries were pooled and adjusted to a final concentration of 50 pm. Template preparation and chip loading were carried out on an Ion Chef™ System (Thermo Fisher). Eight samples were loaded onto an Ion 550™ chip. Finally, Ion 550™ chips were sequenced on an Ion S5™ Sequencer (Thermo Fisher). Analysis of raw sequencing data was performed using torrent suite Software (v5.10.0, Thermo Fisher). For sequencing coverage analysis, the coverageanalysis (v.5.10.0.3) plug‐in was used (Thermo Fisher). Raw reads were aligned to the human reference genome hg19. Variant calling, annotation, and filtering were performed on the Ion Reporter (v5.10) platform using the Oncomine TaqSeq Pan‐Cancer Liquid Biopsy workflow (v5.10). The clinical significance of somatic variants was performed according to Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer.

Genome-wide targeted amplicon amplification was performed using the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo-Fisher, Ottawa, ON, Canada). Total RNA (100 ng) was used to prepare the libraries as per manufacturer’s protocol and described elsewhere [36 (link)]. Prepared libraries were quantified using the Ion Library TaqMan Quantitation kit (Thermo-Fisher, Ottawa, ON, Canada) and then the RNA concentration was adjusted to 100 pM. An Ion Chef instrument (Thermo-Fisher, Ottawa, ON, Canada) was used for loading of Ion 540 chips (Thermo-Fisher, Ottawa, ON, Canada) after which sequencing was performed on an Ion S5 sequencer (Thermo-Fisher, Ottawa, ON, Canada).

The libraries were prepared using the Ion AmpliSeq Library Kit, the Ion AmpliSeq BRCA1/2 Panel, and the Ion Xpress Barcode Adapters Kit, according to the manufacturer's instructions (ThermoFisher Scientific). Sequencing was performed on the Ion Personal Genome Machine (PGM) using the Ion PGM Hi-Q Sequencing Kit and on an Ion S5 sequencer (ThermoFisher Scientific) using the Ion 520 & Ion 530 Kit-Chef. The raw data generated during sequencing was processed using Torrent Server Suite 4.2–5.2 (ThermoFisher Scientific) and the Variant Callerv 4.2–5.2 application. The results were viewed using Integrative Genomics Viewer (IGV; Broad Institute). Additionally, Torrent Server Suite 4.2 generated FASTQ files that were used for analysis by other methods, including the CLC Genomics Workbench, version 7.5.1 (Qiagen) and the Galaxy platform (www.usegalaxy.org). The wANNOVAR program (www.wannovar.usc.edu) was used to annotate the detected variants from Torrent Server Suite and Galaxy. Details are described in S1 Supplementary Methods.

A targeted custom AmpliSeq panel was designed containing coding sequences for three known A1AT deficiency causing genes (SERPINA1, SERPINA3 and ELA2). In total 27 amplicons were designed covering 6.5 kb. The Ion Chef (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to create a next‐generation sequencing library for each sample. These libraries were pooled on an Ion chip and sequenced on an Ion S5 sequencer (Thermo Fisher Scientific). Using a hotspot file on torrent suite 5.4 or 5.8 software (Thermo Fisher Scientific) known variants responsible for A1AT deficiency were called.

Chip loading was carried out using the Ion Chef liquid handling platform (Thermo Fisher Scientific), with 96 NBS2 panel libraries loaded per Ion 540 chip. Seven high throughput sequencing runs were carried out: four on the Ion S5 sequencer and three on the Ion S5 Prime sequencer (Thermo Fisher Scientific). The run plan had the following parameters: analysis parameters: default; reference library: hg19; target regions: NBS2 panel BED file; hotspot regions: none; read length: 200 bp; flows: 500; base calibration mode: default. The plugins used were coverageAnalysis, DataExport and variantCaller.