Ion torrent personal genome machine sequencer

At the time of ultrasound-guided FNA procedure, small portions of the first and second passes were collected for molecular analysis as previously described.^{18 (link)} Total nucleic acids were extracted using Compact MagNA Pure (Roche, Basel, Switzerland). Samples were tested for ThyroSeq v2 using targeted NGS on an Ion 318-chip and an Ion Torrent Personal Genome Machine (PGM) sequencer (Life Technologies, Carlsbad, Calif). For each sample, 2 libraries were created to test for 1) point mutations and small indels and 2) gene fusions and gene expression controls used to estimate the proportion of thyroid follicular cells and other relevant cell types in the collected FNA samples.

For mutation analysis of CYP11B2 positive tumor specimen 1 (B2T1) and CYP11B2 negative tumor specimen 1 (T1) samples, 20 ng of isolated gDNA was used for barcoded library generation by multiplexed PCR using a custom Ion AmpliSeq Panel and the Ion AmpliSeq Library kit 2.0 (Life Technologies, Foster City, CA, USA) according to the manufacturer's instructions. The custom Ion AmpliSeq Panel was designed to target genes previously shown to be mutated in APA or other adrenal hyperplasias/neoplasms. The panel contains 499 independent primer pairs targeting the entire coding regions of genes with reported germline or somatic mutations in PA (KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CACNA1H), genes shown to harbor germline or somatic variants associated with adrenal hyperplasia (phosphodiesterase 11A [PDE11A], phosphodiesterase 8B [PDE8B], protein kinase, cAMP-dependent, regulatory, type 1, α [PRKAR1A], PRAKACA, and armadillo repeat containing 5 [ARMC5]), and oncogene hotspots in guanine nucleotide-binding protein subunit α (GNAS) and catenin, β1, 88kDa (CTNNB1). Template preparation and NGS of multiplexed templates were performed as described previously (11 ) using Ion 318 Chip v2 on the Ion Torrent Personal Genome Machine (PGM) Sequencer (Life Technologies). Data analysis methods for somatic variant identification are described in Supplementary methods.

ZIKV RNA was treated first with DNAse I (Thermo Scientific) followed by cDNA library construction using the Ovation RNA-Seq System V2 (NuGEN) and the Ion Xpress Plus™ gDNA and Amplicon Library Preparation kit (Thermo Scientific). The sheared and purified cDNA library was analyzed using Bioanalyzer (Agilent Technologies). The cDNA template (100 pM) was then prepared and enriched using Ion PGM Hi-Q OT2 kit (Thermo Scientific). To reach a targeted depth coverage of sequence, the enriched ion spheres were then sequenced using the Ion Torrent Personal Genome Machine Sequencer and the Ion PGM Hi-Q Sequencing kit, with a 316 Chip (Thermo Scientific). SMALT (http://www.sanger.ac.uk/science/tools/smalt-0) was used to map the PGM reads to ZIKV genome reference (acc. no. NC_012532). All mapped reads were then filtered using PRINSEQ (http://prinseq.sourceforge.net) for only high-quality reads (mean Q > 30) with length range of 100 – 250 bp, and subjected to 5′/3′ dereplication. The filtered reads were assembled using SPAdes (http://bioinf.spbau.ru/spades) in metagenome mode, and the resulting contig was used as an untrusted contig for the next consecutive SPAdes assembling in single cell and careful mode, which employed mismatch correction using BWA internally.