Hiseq 4000 ngs platforms

Extracted tumor genomic DNA was fragmented into 300 ~ 350 bp using Covaris M220 instrument (Covaris). Sequencing libraries were prepared with KAPA Hyper Prep kit (KAPA Biosystems) with optimized protocols. In brief, cfDNA or sheared tissue DNA were experienced with end-repairing, A-tailing, adapter ligation and size selection using Agencourt AMPure XP beads (Beckman Coulter). Libraries were then subjected to PCR amplification and purification before targeted enrichment.
Indexed DNA libraries were pooled up to 2 µg together with Human cot-1 DNA (Life Technologies) and xGen Universal blocking oligos (Integrated DNA Technologies) as blocking reagents. Customized xGen lockdown probes panel (Integrated DNA Technologies) were used to selectively enrich for 425 or 139 predefined genes. The enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) with 2 × 150 bp pair-end reads to coverage depths of at least 100x, 600x, 5000 × for blood, FFPE, and cfDNA, respectively.

Genomic DNAs from FFPE samples and whole blood control samples were extracted with QIAamp DNA FFPE Tissue Kit and DNeasy Blood and tissue kit (Qiagen), respectively, and quantified by Qubit 3.0 using the dsDNA HS Assay Kit (ThermoFisher Scientific). Library preparations were performed with KAPA Hyper Prep Kit (KAPA Biosystems). A customized panel targeting 425 cancer‐relevant genes was used for hybridization enrichment (Appendix [Link], [Link]). The capture reaction was performed with Dynabeads M‐270 (Life Technologies) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Captured libraries were on‐beads PCR amplified with Illumina p5 (5’ AAT GAT ACG GCG ACC ACC GA 3’) and p7 primers (5’ CAA GCA GAA GAC GGC ATA CGA GAT 3’) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target‐enriched library was then sequenced on HiSeq4000 NGS platforms (Illumina) according to the manufacturer's instructions.

We used the dataset—EGAD00001004164—from the European Genome-phenome Archive, including 34 consecutive patients who underwent GC surgery in 2016 at the AHJU. In brief, total RNA from fresh samples was extracted using Trizol (Invitrogen, USA). Ribosomal RNA was depleted using RNase H followed by library preparation using KAPA Stranded RNA-seq Kit with RiboErase (KAPA Biosystems, USA). The library concentration was determined using KAPA Library Quantification Kit (KAPA Biosystems, USA), and library quality was assessed by the Agilent High Sensitivity DNA kit on Bioanalyzer 2100 (Agilent Technologies, USA), which was then sequenced on Illumina HiSeq4000 NGS platforms (Illumina, USA). Base calling was performed on bcl2fastq v2.16.0.10 (Illumina, USA) for the generation of sequence reads in the FASTQ format (Illumina 1.8+ encoding). Quality control was performed with Trimmomatic (version 0.33). STAR (version 2.5.3a) was used for transcriptome mapping followed by isoform and gene level quantification, as performed using RSEM (version 1.3.0). ACRG and TCGA GC RNA abundance data were downloaded from the NCBI Gene Expression Omnibus (GSE62254) and University of California Santa Cruz (UCSC) Xena platform (https://xenabrowser.net/datapages/), respectively.

cfDNA or fragmented genomic DNA (300~350 bp with Covaris M220 instrument) underwent sequencing library preparation using the KAPA Hyper Prep kit (KAPA Biosystems). In brief, DNA was experienced with end-repairing, A-tailing, adapter ligation, and size selection using Agencourt AMPure XP beads (Beckman Coulter) and then was amplified by PCR and purified.
Indexed DNA libraries were pooled up to 2 µg, together with Human cot-1 DNA (Life Technologies) and xGen Universal blocking oligos (Integrated DNA Technologies) as blocking reagents. A customized xGen lockdown probe panel (Integrated DNA Technologies) covering 425 predefined cancer-related genes was used to perform hybridization capture (Table S1). Enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to target mean coverage depths of at least 100× for WBCs, 1000× for tissue- and bile pellet–derived DNA, and 5,000× for plasma- and bile supernatant–derived cfDNA.

To identify mutation-derived neoantigens, whole exome sequencing was conducted on samples obtained from patients by surgery, biopsy or intravenous blood sampling using Hiseq 4000 NGS platforms (Illumina) with coverage depths of 500x for tumor cells and 100x for blood cells (Novogene Biotech Co., Ltd., Beijing, China) (25 (link)–29 (link)). In addition, formalin-fixed paraffin-embedded (FFPE) samples were used for WES when fresh tumor samples were unavailable.
Bioinformatic analysis was performed by our in-house pipeline iNeo-Suite consisting of multiple modules including sequencing read filtering, genome alignment, mutation calling, HLA typing, MHC affinity prediction, gene expression profiling, vaccine peptide sequence design and mutation-centered prioritization based on therapeutic potency (Supplementary Methods).
Customized clinical-grade long peptides were manufactured through chemical synthesis at GMP-like standard (bacteria-free, > 95.0% purity with endotoxin less than 10 EU/mg) to generate iNeo-Vac-P01. The water solubility of synthesized peptides was tested, and water insoluble peptides were excluded from the final formulation.