Ion ampliseq library kit plus

The samples were chosen for sequencing based on the quantity and quality of viral RNA (Ct < 32 and minimum 7 ng/μL), and the number of viral copies for each sample was evaluated using the TaqMan 2019-nCoV Assay Kit v1 (Applied Biosystems, Waltham, MA, USA).
The viral RNA was reverse-transcribed using the SuperScript™ VILO™ cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA), according to the protocol provided by the manufacturer. The targets for sequencing were obtained using the Ion AmpliSeq™ SARS-CoV-2 Panel (Thermo Fisher, Waltham, MA, USA). Library preparation was performed with Ion AmpliSeq™ Library Kit Plus (Thermo Fisher, Waltham, MA, USA), on an Ion Chef instrument (ThermoFisher Scientific, Waltham, MA, USA). The samples were sequenced on an Ion Gene Studio S5 instrument (Thermo Fisher Scientific, Waltham, MA, USA), using an Ion Torrent 540 chip (Thermo Fisher Scientific, Waltham, MA, USA). The reads were mapped and assembled using the Iterative Refinement Meta Assembler (IRMA). The alignment, lineage, and clade identification were performed using Nextclade and SARS-CoV-2 as a reference genome (MN908947).

NGS analysis was performed on the Ion Gene Studio S5 system (Thermo Fisher Scientific) using two thyroid-specific custom panels, a DNA panel and an RNA panel, as previously described [21 (link), 22 (link)].
Briefly, two libraries were prepared from 15 ng of DNA and 10 ng of RNA with the Ion AmpliSeq™ Library Kit Plus using the IonXpress™ Barcode Adapter 1–96 Kit (Thermo Fisher Scientific). The purified libraries were quantified with Ion Library TaqMan^® Quantitation Kit on the 7900HT Fast Real Time PCR system (Thermo Fisher Scientific). DNA and RNA pooled libraries were clonally amplified on the Ion One Touch2 System and sequenced on Ion Gene Studio S5 (Thermo Fisher Scientific) according to the manufacturer’s instructions and as previously described [23 (link)].
Data analysis was performed using Torrent Suite v.5.10 with Coverage Analysis and Variant Caller plugins and annotated with Ion Reporter 5.12 and the wANNOVAR web server. Gene fusion analysis was performed with Ion Reporter 5.12 software (Thermo Fisher Scientific) using the workflow for gene fusion detection [22 (link)].

Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit on a QIAcube (Qiagen, Valencia, CA, USA). Library preparation was performed using an Ion AmpliSeq Library Kit Plus (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed on an Ion Torrent system (Ion Chef and Ion GeneStudio S5) using an Ion 510 & 520 & 530 Kit—Chef and an Ion 520 Chip Kit (Thermo Fisher Scientific). Sequencing data were mapped using Torrent Suite software (Thermo Fisher Scientific) to human genome hg19, which masked exons 32–44 of TNXB and TNXA sequence by replacing them with “N”s. Single-nucleotide variants (SNVs) and small insertions/deletions were detected from the mapped data using the Torrent Variant Caller plug-in. Copy number variation (CNV) was analyzed using the CNV visualization method for amplification-based NGS data that was established by Nishio et al. (2018) (link). The variants in TNXB were described using the NM_019105.6 transcript reference sequence, and the variant nomenclature was in accordance with the Human Genome Variation Society recommendations.

Libraries were prepared with the Ion AmpliSeq Library Kit Plus according to the manufacturer’s instruction using Ion AmpliSeq SARS-CoV-2 RNA custom primers panel (ID: 05280253, ThermoFisher Scientific). The RNA library preparation included reverse transcription using SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific) with subsequently 16–21 cycles of PCR amplification on Ion Chef. Next-generation sequencing (NGS) reactions were run on Ion Torrent GeneStudio S5 sequencer.
Sequence alignments to the SARS-CoV-2 isolate Wuhan-Hu-1, complete genome (NCBI nucleotide collection, accession number: NC_045512) (Wu et al., 2020 (link)), was performed within the Torrent Server of Ion Torrent S5 sequencer using default settings. The aligned reads were utilized for both reference-guided assemblies. Assembly was performed using the Iterative Refinement Meta-Assembler (IRMA) v.0.6.1 (Shepard et al., 2016 (link)) that produced a consensus sequence for each sample using a >50% cutoff for calling single-nucleotide polymorphisms. IRMA utilizes multiple steps of alignment, variant calling, and consensus building by capitalizing on multiple allele frequency confidence intervals and read depth. Aligned reads were validated through the Integrative Genomics Viewer (IGV) v.2.5.3 (Thorvaldsdóttir et al., 2013 (link)).

Selected FFPE core biopsies (1 mm diameter) of tumor tissue were subjected to DNA and RNA extraction using the AllPrep DNA/RNA FFPE kit (Qiagen). Concentrations were determined with a Qubit 2.0 fluorometer (Life Technologies). 15–20 ng of RNA were reverse transcribed to cDNA (Superscript VILO, Invitrogen). cDNA and 10 ng of DNA were used for library preparation with the Ion AmpliSeq Library Kit Plus with a prostate specific custom multiplex RNA^{32 (link)} panel and barcode incorporation (Ion Torrent, Thermo Fisher). Template preparation of the multiplexed libraries was performed on the Ion Chef system with subsequent sequencing on the Ion S5 XL sequencer (Ion Torrent, Thermo Fisher).
For gene-fusion analysis for each sample all the reads that completely cover the fusion genes specific amplicons from one end to the other end (end-to-end reads) were collected and processed in order to detect the presence of fusions. Fusion genes were called when filtering criteria were met, based on the percentage of the specific end-to-end reads, the number of breakpoint reads and the presence of possible bias toward the forward of the reverse end-to-end reads (Supplementary Table 4; Supplementary Data 5).

NGS libraries were constructed using the Ion AmpliSeq Library Kit Plus (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s standard protocol. Sequencing was performed on the Ion Chef System instrument (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s workflow. A complete library preparation, NGS methodology, and data processing and bioinformatics analysis are described in the SHGP (Cyrus et al., 2018 (link)). Additionally, the Ensembl Variant Effect Predictor (VEP, v104) tool was used to annotate all the identified variants using Ensembl release 100 and assembly GRCh37/hg 19 of the human reference genome (Lee et al., 2007 (link)). Variants were also compared against the Genome Aggregation Database (GenomAD). Functional assessment of the identified variants was conducted using SIFT (v5.2.2), PolyPhen-2 (v2.2.2), and CADD (v1.6) (Cyrus et al., 2018 (link); O'Seaghdha et al., 2014 (link); Santín et al., 2011 (link)).