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.