Tumour specimen collection and nucleic acid extractions
All participants gave their written informed consent to participate in this study which has been approved by the local ethical review board (approval code: 2016/2-31/1). Fresh frozen tumour tissue was isolated from surgical specimens by a clinical pathologist at the Karolinska University Hospital, Stockholm, Sweden. Tumour tissue was snap frozen and tumour representativity was confirmed using routine haematoxylin-eosin (HE)-stained sections, followed by a visual estimation of tumour cell proportion. Nucleic acids were manually extracted from tumour tissue using the DNeasy and RNeasy kits (Qiagen, Hilden, Germany) at the Clinical Molecular Pathology laboratory using accredited protocols (SWEDAC). DNA and RNA isolates were quantified using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MS, USA) and quality controlled using a 4200 Tapestation (Agilent Technologies, Santa Clara, CA, USA). Germline DNA was extracted from white blood cells using EZ1 DNA Tissue Kit (Qiagen) and quantified using Qubit.
Whole genome sequencing
DNA from fresh-frozen tumour samples and white blood cells was converted to sequencing libraries using a PCR-free paired-end protocol (Illumina TruSeq, San Diego, CA, USA) and sequenced on the NovaSeq 6000 (Illumina) aiming at 30x coverage. The median coverage for tumour DNA was 26 – 36x (330 – 491 M read pairs) and 30 – 38 x for germline DNA.
Whole transcriptome sequencing/Preparation of RNA libraries
RNA-seq was performed on total RNA from fresh frozen tumour tissue. Sequencing libraries were generated using PCR-free paired-end protocol (Illumina TruSeq, San Diego, CA, USA) and sequenced on the NovaSeq 6000 (Illumina) platform aiming for at least 20 M reads per sample. Raw FASTQ reads were submitted for fusion gene analysis as described below.
SNVs were called using Balsamic (https://github.com/Clinical-Genomics/BALSAMIC/), which uses Vardict, TNscope and TNHaplotyper for calling SNvs and small indels and were visualised in SCOUT - a custom-developed decision support system.
The four following criteria were applied for manual filtering of variants identified in by the bioinformatic caller: 1) variants should be located in the exonic or splice regions, 2) >5% allele frequency in the tumour sample, 3) <0.001% allele frequency in the normal sample, and 4) frequency in the The Genome Aggregation Database (gnomAD, v.2.1.1) < 0.01. Next all variants were manually inspected using the Integrative Genomics Viewer  and functionally interpreted. All prefiltered variants were also submitted to the Molecular Board Portal Karolinska  for functional annotation. Filtered variant lists are available in the supplementary data.
We used FusionCatcher (version 1.20 with standard settings ) to screen the transcriptome for fusion gene transcripts in all cases. Putative in-frame fusion transcripts were filtered for blacklisted and banned variants, genes with common mapping reads, adjacent genes as well as limited anchorage sequences (short reads). Lastly, all fusion transcripts were compared to rearrangements identified in the WGS data. SVs from WGS data were identified using the FindSV pipeline (https://github.com/J35P312/FindSV) incorporated into MIP (https://mip-api.readthedocs.io/en/latest/) and visualised in the SCOUT interface (https://github.com/Clinical-Genomics/scout).
Copy Number Alterations
Copy number alterations were detected using the FindSV pipeline. FindSV performs variant detection using CNVnator V0.3.2 using 1kb bins  and TIDDIT V2.0.0 ; as well as annotation using the variant effect predictor 92 (McLaren et al. 2016) and SVDB . Next, the variants were visualized using IGV  and VCf2cytosure . In addition, copy number analysis was performed and Manhattan plots were generated with CNVPytor.1 Samples were analysed with the read depth option and with a bin size of 100kb . Selected CNVs were also verified in the SCOUT interface (https://github.com/Clinical-Genomics/scout).
Isolation of cfDNA
Peripheral blood samples were collected from all patients before surgery (24 - 0 days prior) in Streck Cell-Free DNA BCT tubes and stored in room temperature for a maximum of 5 days before plasma isolation. Blood volumes ranged from 6-20 ml/patient. After centrifugation at 1600 x g. (10 min 4°) the plasma supernatant was removed and centrifuged at 16 000 x g. (10 min 4°). Cell-free plasma volumes ranged from 3-9 ml/patient and were frozen at -80°C in cryovials. Plasma from blood donors was collected as reference plasma.
Samples were thawed in room temperature and cfDNA extraction was performed on 3 ml aliquots of plasma (one or multiple per patient depending on total plasma volume) using QiAamp Circulating Nucleic Acid Kit (Qiagen, Manchester, UK). cfDNA was eluted in 40 µL AVE buffer and stored in –20°C.
ddPCR design and analysis
Custom ddPCR assays containing sequence-specific primers and FAM/HEX labelled probes were designed based on WGS results according to the Rare Mutation Detection Best Practices Guidelines (BioRad, Hercules, California, USA) and ordered from Integrated DNA Technologies IDT (Coralville, Iowa, USA). For SNV assays, the HEX-probe was designed for the wild type allele. For the SV assay, a commercial non-mutated reference gene was used (ABCC9). Amplicon lengths were 65-91 bp. For sequence details see Supplementary table 1. All assays were tested on a gradient-ddPCR to define optimal annealing temperature. Gradients were run on normal control gDNA (10 ng/well), NTC and positive control (1ng patient tumour DNA in a background of 9 ng normal control gDNA). No false positives were observed.
Before ddPCR analysis, all cfDNA aliquots from the same patient were pooled. ddPCR reaction mixes were then prepared with 10 µL 2x ddPCR Supermix for Probes (No dUTP, BioRad), 11 µL cfDNA sample and 1 ul custom assay containing (final primer/probe concentration of 900 nM/250 nM respectively). Reactions were run in triplicates alongside triplicates of non-template, wild type (10 ng gDNA) and positive controls (1 ng gDNA from tumour in 9 ng wt gDNA). Additionally, a minimum of 9 wells of cfDNA from healthy plasma donors were run as background control. ddPCR reactions were run on the QX200 AutoDG Droplet Digital PCR System and QX200 Droplet Reader (BioRad) according to the manufacturer’s instructions. The following cycling conditions were used: 1 cycle at 95°C for 10 min, 40 cycles at 94°C for 30 s and custom annealing temperatures* for 1 min, 1 cycle at 98°C for 10 min, and 1 cycle at 8°C infinite, all at a ramp rate of 2°C/s.
Data was analyzed using the QuantaSoftPro Software (BioRad) and results were manually reviewed. The threshold for positive droplets was based on the control samples run together with patient samples in each assay.