Ion torrent s5xl platform

Genomic DNA was extracted from peripheral blood samples taken from patients. TGS was performed for 10 of the 15 patients with the Ion Torrent S5XL platform (Thermo Fisher Scientific Inc., Waltham, MA, USA) using a panel consisting of 88 genes associated with RP, and for further two patients with the Illumina NextSeq platform (Illumina Inc., San Diego, CA, USA) using the TruSight One Sequencing Panel. For the remaining three patients, WES was performed. All exons of all genes (approximately 22,000) were captured using a SureSelect kit (Version C2; Agilent Technologies Inc., Santa Clara, CA, USA). The captured genomic regions were sequenced using a NovaSeq platform (Illumina Inc., San Diego, CA, USA). Analysis of raw genome sequencing data included alignment to the reference sequence (NCBI genome assembly GRCh37; accessed in February 2009). For TGS, mean depth of coverage was approximately 500-fold, with 99.2% coverage higher than 20-fold. Variant calling, annotation, and prioritization were performed as previously described³² . The requirement for verification of identified variants was waived for Torrent S5XL sequencing data when the read depth was over 100 reads and the allele frequency was 40–60%^{33 (link)}, while validation of Illumina NextSeq and WES data was achieved with subsequent Sanger sequencing.

The Oncomine Comprehensive Assay v3 (OCAv3) (ThermoFisher Scientific) was performed on the IonTorrent™ S5 XL platform, following manufacturer protocols (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0015885_OncomineComprehensiveAssay_v3_UG.pdfLast downloaded 12/27/2019). OCAv3 is an amplicon-based targeted assay that enables the detection of relevant SNVs, amplifications, gene fusions, and indels from 161 unique genes (Supplementary Table 1).

Bacterial DNA was extracted and whole genomes were sequenced by Illumina HiSeq X platform at SciLifeLab (Stockholm, Sweden) and Ion Torrent S5 XL platform (Thermo Fisher Scientific, Waltham, Massachusetts, US) at The Public Health Agency of Sweden as previously described [26 ]. The Illumina sequencing reads were de novo assembled with SKESA (version 2.3.0) [27 (link)]. The Ion Torrent sequencing reads were de novo assembled with SPAdes (version 3.12.0) in “careful mode” [28 (link)]. The genome assemblies were annotated with Prokka (version 1.14.6) [29 (link)]. The assemblies of all strains in this study were deposited in GenBank with accession numbers and metadata shown in Table S1.