GSM5016463: HCC827 EGFR M-RT; Homo sapiens; OTHER
DNA was extracted using the Qiagen Dneasy kit Genomic DNA was isolated (Qiagen DNeasy Blood & Tissue Kit, catalog # 69504) from HCC827 cells transduced at an MOI of approximately .3 with EGFRdel746-750 cDNA that was packaged using either the WT-RT or M-RT and otherwise identical conditions. Two microgram of this DNA was used for two PCR cycles (using New England Biolabs Q5 Hot Start Polymerase, catalog # M0494S) of a 243 bp amplicon of the EGFR open reading frame using the primers TTGCGGTGTCAATCTTACCANNNNNNNNCAGCGCTACCTTGTCATTCA and ATGAATCCAAACCGGATGAANNNNNNNNTTGCAGCCCATTTCTATCAA to label both ends of individual amplicon molecules with unique molecular identifiers (UMIs) of 8 nucleotides. After removal of the UMI-containing primers by Exonuclease I (NEB) digestion and column purification (Qiagen QIAquick PCR purification kit catalog # 28104), the UMI-tagged amplicons were then further amplified using the primers TTGCGGTGTCAATCTTACCA and ATGAATCCAAACCGGATGAA and purified (Qiagen QIAquick PCR purification kit catalog # 28104) to generate 299 bp products. For these, barcoded Illumina libraries were prepared with the KAPA HyperPrep Kit (Roche) for sequencing on the NextSeq500 (Illumina) with V2.5 150PE chemistry. PE reads were demultiplexed and from each read in the fastq file, 20bp primer was trimmed from the 5' end and 8bp was extracted which contained the UMI information. We used UMIs in which all basepairs had a sequence quality value of >=30 for further analysis. The UMI combinations from plus and minus reads were quantified and assigned a UMI combination and its count to each readID. After preprocessing (including further trimming of the UMI and the sequences of the second primers), singletons from preprocessed fastq files were separated using fastq-pair tool. Resulting fastq files were mapped to the EGFR cDNA sequence using BOWTIE2 (v.2.3.5). To identify mismatches in each mapped sequence we used samtools calmd command and custom in-house python and perl scripts. First, for each read we identified mismatches in each position and combined this information with UMI counts. Then, for each base pair mismatch, we calculated the ratio between the total number of reads that show a given mismatch/ and the total number of reads sequenced (given by total number of UMIs). Events with greater than .9 mismatch rate were used for further analysis.