Nextseq500 apparatus

50–100 ng/sample RNA from each animal was used as input for cDNA synthesis and amplification with the SMARTer Ultra Low Input RNA kit (Clontech, CA) following manufacturer recommendations with 13 cycles of PCR amplification. cDNA quantity and quality were evaluated with a Qubit fluorometer and TapeStation 4200, respectively.
100pg of amplified cDNA was used to generate DNA libraries using Illumina Nextera XT DNA Library Kit (Illumina, CA). Twelve library amplification cycles were applied per manufacturer’s recommendation. Library quantity and quality were evaluated as described above for the cDNA. Illumina paired-end 151 bp sequencing was performed on a Next-seq 500 apparatus (Illumina, USA).

The bacterial genomes used in this study are presented in Table 1. Total bacterial DNA was extracted from pure bacterial cultures using the Wizard genomic DNA purification kit (Promega), following the manufacturer’s protocol.
DNA samples of IPO strains were sequenced using short-read sequencing (Illumina, San Diego, CA, USA). Random sheared shotgun library preparation was performed using the Truseq Nano DNA Library Prep kit (dual indexing) following the manufacturer’s protocol. The samples were loaded on a paired-end flowcell using the Hiseq PE cluster kit V4 (Illumina). One hundred twenty-five bp paired-end sequences were generated on a Hiseq 2500 (Illumina, San Diego, CA, USA).
Genome sequencing of CH and CFBP strains was performed at the next-generation sequencing core facilities of the Institute for Integrative Biology of the Cell (Avenue de la Terrasse 91190 Gif-sur-Yvette France). Nextera DNA libraries were prepared from 50 ng of high-quality genomic DNA. Paired-end 2 × 75 bp sequencing was performed on an Illumina NextSeq500 apparatus with a High Output 150 cycle kit. CLC Genomics Workbench (Version 9.5.2, Qiagen Bioinformatics) was used to assemble reads. Final sequencing coverage was between 49 and 79. Coding sequences were predicted using the RAST server [25 (link)] with the Glimmer 3 prediction tool [26 (link)].

Tumor areas were determined by a certified pathologist and were manually macro-dissected from the FFPE tissues. RNA was extracted using an All Prep DNA/RNA/miRNA Universal kit (Qiagen, Belgium) according to the manufacturer’s protocol. The RNA quality (N = 36) was assessed using a BioAnalyzer (Agilent, Belgium), and the proportion of RNA with a length higher than 200 bases (DV200) was measured. Only 19 out of 36 met a suitable RNA quality, allowing for sequencing. TruSeq® RNA Access Library Prep Kit (Cat. No. RS-301-2001 and RS-301-2002) (Illumina, The Netherlands) was used to prepare libraries, and next-generation sequencing was performed on a NextSeq500 apparatus (Illumina, The Netherlands), in paired-end 2 × 75 bp high output mode.
We performed a series of transcriptome computational analyses to better understand patient heterogeneity between LT and ST survivors. After quality control and adaptor trimming with Trimmomatic^{75 (link)}, sequence data were mapped to the Genome Reference Consortium GRCh38 assembly using STAR v2.5.2^{76 (link)}. Read counts for known genes were generated using the function HTSeq-count v0.6.1p^{77 (link)} and data were normalized in DESeq2 v1.20.0^{78 (link)} as shown in Fig. 1. The study’s analytic workflow is depicted in Fig. 1A–L.