Ion s5 xl system

The procedure of study material preparation and sequencing was conducted as previously described in [28 (link)].
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood specimens using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). Proportions of white blood cells subpopulations in AAA group were obtained from venous blood morphology analysis results and were presented in Figure S1.
Small RNA fractions (for miRNA expression analysis) were isolated from PBMCs specimens of twenty eight AAA patients and nineteen control subjects using MirVana microRNA Isolation Kit (Ambion, Austin, TX, USA).
Total RNA specimens (for transcriptome analysis) were isolated from PBMCs samples of seven randomly selected AAA patients and seven randomly selected controls using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA).
Small RNA and transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit, Ion Xpress RNA-Seq Barcode 01-16 Kit and sequenced on Ion 540 chips (all Life Technologies, Carlsbad, CA, USA) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences of small RNA and transcriptomic libraries were aligned to 2792 human miRNAs from miRBase v21 (http://www.mirbase.org) and to 55,765 genes of hg19 human genome, respectively.

The total microbial DNA was obtained from the colon content or fecal pellets by E.Z.N.A.^® Stool DNA Kit (OMEGA, Norcross, GA, USA) and quantified by NanoDrop™ 2000 Spectrophotometer (ThermoFisher, Waltham, MA, USA). PCR amplification of V3–V4 regions of bacterial 16S rRNA genes was performed using universal primer sequences 341F (5’-barcode-CCTAYGGGRBGCASCAG) and 806R (5’-barcode-GGACTACNNGGGTATCTAAT). The PCR products were purified by QIAquick Gel Extraction Kit (Qiagen, Germantown, MD, USA). Thereafter, the amplicon libraries were prepared using Ion Plus Fragment Library Kit (ThermoFisher, Waltham, MA, USA) and the sequencing was performed on Ion S5™ XL system (ThermoFisher, Waltham, MA, USA).

RNA sequencing was carried out on the indicated samples using a targeted AmpliSeq Transcriptome panel on Ion S5 XL System (ThermoFisher Scientific, Cambridge, MA, USA). In brief, ~30 ng of Turbo DNase treated RNA was used for cDNA synthesis using a SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific) followed by amplification using Ion AmpliSeq gene expression core panel primers. The enzymatic shearing was performed using FuPa reagent to obtain amplicons of ~200 bp and the sheared amplicons were ligated with the adapter and the unique barcodes. The prepared library was purified using Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN, USA) and the purified library was quantified using an Ion Library TaqMan™ Quantitation Kit (Applied Biosystems, Waltham, MA, USA). The libraries were further diluted to 100 pM and pooled equally with four individual samples per pool. The pooled libraries were amplified using emulsion PCR on Ion OneTouch™ 2 instrument (OT2) and the enrichment was performed on Ion OneTouch™ ES following the manufacturer’s instructions. Thus, prepared template libraries were then sequenced with Ion S5 XL Semiconductor sequencer (ThermoFisher Scientific, Cambridge, MA, USA) using the Ion 540™ Chip.

The DNA was amplified using random PCR as described elsewhere [14 (link)]. The PCR tag-sequence was cleaved away using EcoRV (Thermo Fisher, Waltham, MA, USA). The RNA was reverse-transcribed into cDNA and amplified by single-primer isothermal amplification (SPIA) using the Ovation RNA-Seq V2 kit (NuGEN, San Carlos, CA, USA) according to the manufacturer’s instructions. The random PCR products and the SPIA products were then purified using the GeneJET PCR purification kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. The concentrations of the purified random PCR products ranged from 18 to 25 ng/µL, and those of the purified SPIA products ranged from 36 to 66 ng/µL. All the products were sequenced at SciLifeLab/Genome Center (Uppsala, Sweden) using the Ion S5 XL system (Thermo Fisher, Waltham, MA, USA) and two 530 chips. The main type of sequencing errors associated with this platform are insertions and deletions (indels).

DNA of the primary tumor and adjacent non-cancerous liver was extracted from ten 5 μm-thick slides made from formalin-fixed paraffin-embedded (FFPE) samples acquired at baseline using the Maxwell RSC DNA FFPE Kit (Promega, Madison, US). The quantity and quality of FFPE-derived DNA samples were checked using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, US). DNA was fragmented and used for library construction according to the manufacturer’s instructions. The 18 genes including TERT promoter (chr5, 1295151-1295313), CTNNB1, TP53, ARID1A, ARID2, ALB, AXIN1, APOB, CDKN2A, RPS6KA3, FGFR1, FGFR2, FGF3, FGF4, FGF19, CCND1, ATM, and APC were enriched using the Ion AmpliSeq Custom Panel (Thermo Fisher Scientific). Sequencing was performed with paired-end reads on the Ion S5 XL system (Thermo Fisher Scientific). Sequencing reads were aligned to the hg19/GRCh37 reference sequence and analyzed using Ion Reporter Software (Torrent Suite Software; Thermo Fisher Scientific). Called variants were considered germline mutations if called in the control non-cancerous tissue or found in the dbSNP^{12 (link)} database and 1000 Genomes Project^{13 (link)}.

Whole-genome amplification of SARS-CoV-2 virus genome was performed using ARTIC primer V4 with reverse transcription polymerase chain reaction using BioMaster RT-PCR—Premium (Biolabmix, catalog number RM05-200, Russia). DNA libraries were constructed using NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. DNA sequencing was performed using Ion 540 Chip and Ion S5XL System (Thermo Fisher Scientific, Waltham, MA, USA).