Targeted Next generation sequencing for gene mutations
One representative and cellular FNA smear was photographed for documentation and the entire smear was scraped off the slides using a surgical blade manually. DNA was extracted using the Qiagen DNeasy Blood and Tissue Mini kit (Qiagen,Gmbh, Germany) and quantified by using Qubit ds DNA HS Assay kit (Thermo Fischer Scientific, Oregon). Only samples with a minimum DNA concentration of 2ng/μl were selected for NGS. An amplicon library was generated from 30-50ng/ μl of DNA from each sample using the Ion Ampliseq Cancer Hotspot Panel version 2.0 (Life Technologies and  Ion AmpliSeq™ Library Kit 2.0 (Ion Torrent, USA) which evaluates hotspots in 50 genes including ALK, IDH1, TP53 and others (supplementary table 1). Following PCR amplification of target sequences, barcodes were ligated to the amplicons using the Ion Xpress Barcode Adaptors Kit (Life Technologies). 100pM of DNA library of each sample was taken, pooled and run using Ion PGM Hi-Q OT2 kit. Emulsion PCR was performed manually using the Ion Xpress Template Kit (Life Technologies) followed by manual breaking and isolation of ion spheres (ISP). Selective ISPs with DNA were isolated and sequenced on an Ion 318 chip using Ion PGM Hi Q Sequencing kit and Ion S5 sequencer. Appropriate quality control parameters were assured with at least 300000 reads with a quality score of AQ20; minimum coverage of 250X and an allele frequency cut-off of 5%. The data files were analyzed by Torrent Suite software V2.0.1 (Life Technologies) using Hg19 as the reference; variants were detected using the Torrent Variant Caller software V1.0 (Life technologies) and visualized by Integrative Genomics viewer. Any variant observed was checked in the COSMIC and ClinVar databases and classified as per ACMG guidelines as benign, variant of uncertain significance (VUS), likely pathogenic and pathogenic.