AMPLICON of human: adult bone_marrow tumor: Sample H_OO-10DD-1067-1067_day_0_marrow
In conjunction with the AmpliSeq Library Kit 2.0, 50ng of input DNA in 6ul was combined with 10ul of a custom AmpliSeq primer pool covering exons 1 and 4-10 of TP53, as well as the sites of commonmutations in DNMT3A (R882), IDH1 (R132), U2AF1 (S34), IDH2 (R172 and R140), SRSF2 (P95), and ASXL1. It also targets 30 additional sites on chromosome 17 that can be used to measure loss of heterozygosity. It was also combined with 4ul of the 5X Ion AmpliSeq HiFi Mix. All reactions were cycled: 99 degrees C for 2 minutes followed by 20 cycles of 99 degrees C for 15 seconds and 60 degrees C for 4 minutes. After amplification, primer sequences were digested by adding 2ul of FuPa Reagent and cycling at 50 degrees C for 10 minutes, 55 degrees C for 10 minutes, and 60 degrees C for 20 minutes. Adapters were ligated onto the samples through the addition of 4ul of Switch Solution, 2ul of Ion Xpress Barcode adapter mix, and 2ul of DNA Ligase. Samples were purified using 45ul of Ampure XP beads (Agencourt/Beckman Coulter). The libraries were quantified through use of the KAPA Library Quantification Kit for Ion Torrent and diluted to 100pM with Ion Torrent Low TE. Dilutions of anywhere from 20-24 libraries were then pooled together. Template preparation was carried out on the Ion OneTouch 2 instrument in conjunction with the Ion Personal Genome Machine (PGM) Hi-Q OT2 Template Prep Kit according to revision A of the associated protocol (publication part number MAN0010902). The 400bp workflow was selected. The amplification reaction consisted of 25ul of nuclease-free water, 800ul of Ion PGM Hi-Q Reagent Mix, 50ul of Ion PGM Hi-Q Enzyme Mix, 25ul of the pooled library diluted to 8pM, and 100ul of Ion PGM HiQ Ion Sphere Particles (ISPs). Following automated template prep on the OneTouch 2, enrichment of template-positive ISPs was done on the Ion OneTouch ES instrument. The Ion PGM Hi-Q Sequencing Kit was used for sequencing on the Ion Torrent PGM according to the revision D.0 of the manufacturer's protocol (publication part number MAN0009816). Each library pool was sequenced on a single Ion 318 Chip Kit (v2) with400bp read length settings (850 flows).