Miseq v2

Genomic DNA was sheared, converted into truncated Illumina libraries and enriched (via hybridisation to 5’ biotinylated 120-mer DNA oligonucleotides (xGen Lockdown Probes) as described by Bardan (2019) [13 ]. A total of 125 nuclear SNPs (67 ancestry-informative; Table 1, 23 phenotypic; Table 2, and 35 Y-chromosome; Table 3, with one SNP shared between ancestry and phenotype) were include in the bait set. The 124 SNPs provide broad categorisation of continental biogeographic ancestry (African, European, Asian, Native American and Oceanian), major Y-chromosome haplogroups and hair/eye colour prediction, and were developed as a customisable panel for forensic intelligence gathering (Bardan 2019).
Enriched DNA for all 28 samples were combined into a single pool at 5nM concentration prior to paired end sequencing using Illumina MiSeq V2 with read length 2x150 base-pairs (300 cycles).

Targeted DNA sequencing libraries were constructed using SureSelect^QXT Reagent Kit (Agilent Technologies, Santa Clara, CA) with 50 ng of genomic DNA. Briefly, tumor DNA was enzymatically fragmented and tagged to generate adapter-tagged libraries. Biotin-labeled probes specific to the targeted regions of interest (ROI) via hybridization, and libraries were enriched for ROI using streptavidin beads, then amplified, dual-indexed, and pooled for sequencing; quality of the libraries were measured with 2200 TapeStation (Agilent) and quantified using Qubit 2.0 (ThermoFisher Scientific, Waltham, MA). Targeted RNA sequencing (Fusion Panel) libraries were prepared using Archer™ Universal RNA Reagent Kit v2 for Illumina with 150 ng of input RNA or total nucleic acid (TNA) (ArcherDX, Boulder, CO). Target-enriched library was generated by using a combination of gene-specific primers and universal adapters with minor modification to the manufacturer’s instruction (ArcherDX). Briefly, RNA was reverse-transcribed to generate cDNA and molecular barcode adapters were ligated to cDNA followed by two rounds of target-specific PCR. The library was quantified using KAPA Biosystems qPCR kit (KAPA Biosystems, Wilmington, MA). All libraries were sequenced on Illumina MiSeq or HiSeq platform with MiSeq v2 or HiSeq Rapid SBS v2 300 cycle reagent kit (Illumina, San Diego, CA).

NGS libraries were prepared as described previously [22 (link),23 (link)]. Briefly, 100 ng of fragmented genomic DNA was ligated with a specific barcode and pooled with 23 other sample libraries for a 24-plex run that was captured using the SeqCap EZ Choice Library system according to the manufacturer’s protocol (Roche NimbleGen, Inc., Madison, WI, USA). Captured libraries were diluted to 8 pM or 1.3 pM for sequencing with the MiSeq v2 or NextSeq v2.5 mid output kits, respectively (Illumina, San Diego, CA, USA). Sequencing reads were generated as 2 × 150 bp paired-end reads with post-sequencing file conversion to FASTQ for alignment with NextGene software version 2.4.2.3 (SoftGenetics, LLC, State College, PA, USA) using standard alignment settings. Variants were filtered at an allelic fraction of > 10% to minimise the impact of sequence artifacts and mutational burden and were classified by a clinical molecular geneticist based on the College of American Pathologists (CAP) and the American College of Medical Genetics and Genomics (ACMG) standards and guidelines for pathogenicity [24 (link),25 (link)]. For this study, all assessed Tier I/II variants (variants of strong and potential clinical significance (therapeutic, prognostic and diagnostic)) [24 (link)] and ACMG 1/2 variants (pathogenic or likely pathogenic variants) [25 (link)] were reported.

Bacterial DNA was extracted from sweat samples by Sambrook’s Phenol: chloroform: isoamyl alcohol method (Sambrook, Fritsch & Maniatis, 1989 ). Extracted bacterial DNA samples were measured on the Nanodrop spectrophotometer at 260 nm and 280 nm absorbance. Further, these DNA samples were diluted to 25 ng/ul, of which 5 ng/ul was used for 16S rRNA sequencing of V3 and V4 region on Illumina miSeq V2 standard flow cell technology.

DNA from PDO cells was extracted using the All Prep DNA/RNA Mini Kit (Qiagen, Germany). Library preparation was performed using a QIASeq™ custom targeted thirty gene DNA panel (Qiagen, Germany), detailed in Supplementary Table 1. Sequencing was performed using a MiSeq v2 (Illumina, USA), 300 cycle flow cell paired end at 500X coverage on a MiSeq™ (Illumina, USA) next generation sequencing (NGS) platform.