GSM5016534: H358 KRAS-WT + dox rep 1; Homo sapiens; OTHER
Qiagen Dneasy Blood and Tissue kit For EGFR the entire open reading frame, for BCR-ABL two large amplicons, for RUVBL1 three short barcoded amplicons, and for KRASwt/KRASG12C two short barcoded amplicons were then PCR amplified, purified (Qiagen QIAquick PCR purification kit catalog # 28104), ran on a D5000 HS ScreenTape on the Agilent Tapestation 4200 and DNA concentrations determined by Qubit. For EGFR and BCR-ABL the PCR products were sheared on the Covaris S2 for 40 seconds and libraries are prepared with the KAPA Hyper Prep Kit. Samples were end repaired, 3' ends were adenylated and then barcoded with multiplex adapters. Samples are size selected prior to PCR to remove small fragments. The short RUVBL1 and KRASwt/KRASG12C PCR products were directly (i.e. without sonication) ligated with the adapters. Four cycles of PCR were used to amplify the libraries, which were then purified with AmpureXP beads and validated on the Agilent Tapestation 4200. Before being normalized and pooled, samples were quantified by Qubit then ran using the Nextseq500 V2.5 150PE or MiSeq V2.5 250/300PE chemistry. Reads obtained from pooled samples were separated into sample specific readIDs based on the barcode information. For each read in the pooled sample fastq file, the barcode and primer sequences were trimmed from the 5' end. Using the trimmed fastq file and readIDs obtained in the first step, sample specific fastq files were generated using BBMap (v 38.11). These reads were mapped to the reference cDNA sequence using BWA (v. 0.7.15). For identification of point mutations, read pileup information was obtained from mapped reads using the samtools (v.0.1.19) mpileup command. From the read pileup information, per base mismatch/indel information was obtained using pileup2base tool. Per base error ratios were computed using an in-house Perl script, and the frequency of mutations at positions was compared between the parental (no doxycycline, no drug) and +doxycycline/drug surviving cells. For identification of deletions, the trimmed reads were mapped to the reference cDNA sequences using speedseq, which in turn uses BWA-mem algorithm to map the reads. In addition to the alignment, speedseq tool marks duplicates and identifies split and discordant read pairs. The speedseq mapped files were used to call structural variations (SVs) using Lumpy SV caller. Lumpy SV calls were further filtered using number of split and discordant supporting reads.