Ion torrent pgm next generation sequencing

A number of 22 bladder cancer samples, the same ones analyzed by microarray (except one samples from microarray patien cohort with low DNA concentration), were sequenced using Ion Ampliseq Cancer Panel and Ion Torrent PGM Next Generation Sequencing (Thermo Fischer Scientific); this panel contains the most relevant hot spot mutation. The amplicon libraries were prepared with 20 ng of DNA and the Ion Ampliseq™ Library Kit 2.0 (Life Technologies) and this was followed by a purification step using AMpure XP Beads (Beckman Coulter). Lastly, Qubit 2.0 was used for the quantification using Qubit HS DNA kit. For sequencing, four bar-coded 100pM-diluted libraries were used for each Ion 316 Chip (Thermo Fischer Scientific). Ion Torrent PGM Machine (Thermo Fischer Scientific) performed the sequencing, using the Ion PGM HI-Q Sequencing 200 kit. The software Torrent Suit 5.6 and Ion Reporter 5.6 executed the bioinformatics analysis, specifically for data trimming alignment and variant calling.

We explored linkage disequilibrium (LD) structure around the target SNP rs1413299 using the HapMap Project database (Release 3 version 27, CHB+JPT) by Haploview (Barrett et al., 2005 (link)). A total of 1,039 variants (total bases: 130 kb, overall coverage: 96.9%) were successfully designed within the LD block interval at 9q22.33 (chr9:101,730,000-101,860,000, hg19). Sequencing was conducted using Ion Torrent PGM Next Generation Sequencing (Thermo Fischer Scientific, Waltham, MA, USA) and four variants with minor allele frequency (MAF) >5% and in high LD with the target SNP rs1413299 (r²>0.8) were selected. In order to identify additional new variants, we also included SNPs with P < 1 ×10⁻³ in the 1Mb region surrounding rs1413299 from previous GWAS results and the SNPs with high LD (r²>0.7) to rs1413299 from SNAP (https://www.broadinstitute.org/snap/snap). Finally, ten variants were included for the subsequence validation study.
Genotyping of the ten SNPs (rs10819587, rs10988451, rs1413298, rs1572136, rs1889268, rs4743305, rs7021675, rs7027650, rs7031588, and rs73503719) was performed using the iPlex MassARRAY platform (Sequenom, Inc.) according to manufacturer’s protocols.