A flowchart describing the analyses, steps and number of samples used in each individual section can be found in Supplementary Fig. 5.
Patient recruitment and sample collection
The protocol for sample collection was approved by the Mexican National Cancer Institute’s (Instituto Nacional de Cancerología, INCan, México) Ethics and Research committees (017/041/PBI;CEI/1209/17) and the United Kingdom’s National Health Service (18/EE/00076). Patient samples collected for the Utah cohort analysis were derived as described previously47.
Recruitment of patients and sample collection took place from 2017 to 2019. Patients attending follow-up appointments at INCan who had previously been diagnosed with acral melanoma were offered the chance to participate in this study and, upon signing a written consent form, were asked to provide access to a FFPE sample of their tumour tissue that had been kept at the INCan tumour bank, as well as a saliva or normal adjacent tissue sample. Patients provided samples and their clinical data in Excel format with written informed consent. FFPE samples underwent inspection by a medical pathologist to establish whether sufficient tumour tissue was available for exome sequencing. Saliva samples were collected using the oragene DNA kit (DNAGenotek, catalogue no. OG-500).
DNA and RNA extraction
DNA extraction from all saliva samples was performed at the International Laboratory for Human Genome Research from the National Autonomous University of México (LIIGH-UNAM) using the reagent prepITL2P (DNAGenotek, catalogue no. PT-L2P) and the All-Prep DNA/RNA/miRNA Universal Kit (Qiagen, catalogue no. 80224). DNA and RNA extraction from FFPE samples was performed at the Wellcome Sanger Institute (UK) using the All-prep DNA/RNA FFPE Qiagen kit. Samples with >0.1 ng μl−1 were sequenced through the Sanger Institute’s standard pipeline. Saliva and adjacent tissue samples were used for whole-exome sequencing, and only saliva samples were used for genotyping.
Genotyping
Genotyping was performed using Illumina’s Infinium Multi-Ethnic AMR/AFR-8 v.1.0 array at King’s College London and Infinium Global Screening Array v.3.0 at University College London. Sufficient germline DNA was available for genotyping for 80 out of 92 samples (86.9%). Ancestry estimation was performed using PLINK v.1.9, and ADMIXTURE48 v.1.3.0 for unsupervised analysis together with the superpopulations of the 1000 Genomes dataset49. Five superpopulations were identified, corresponding to AFR (Q1), AMR (Q2), SAS (Q3), EAS (Q4) and EUR (Q5) (Supplementary Table 2 and Supplementary Fig. 1).
Exome sequencing and data quality control
FFPE samples, saliva and normal adjacent tissue underwent whole-exome sequencing as follows: Exome capture was performed using Agilent SureSelect AllExon v.5 probes and paired-end sequencing was performed at the Wellcome Sanger Institute (UK) in Illumina HiSeq 4000 machines. Control and tumour samples were sequenced to a mean coverage of 43.72×. Alignment was done using BWA-mem50 v.0.7.17, using the GRCh38 reference genome. Sequencing quality filters were performed using Samtools v.1.9 stats51 and fastqc v.0.11.3 (ref. 52). Sample contamination was estimated using the GATK v.4.2.3.0 tool CalculateContamination53. Concordance between sample pairs was estimated using the Conpair v.0.2 tool54. Samples that had less than 90% similarity with their pair (tumour-normal) or showed a level of contamination above 5% were excluded from the study. After this step, 123 samples remained for further analysis.
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