Study uncovers role of ultraviolet radiation in development of rare leukemia in the skin danafarber nature
). We specifically considered loci of which only a single allele is present in the genomes of healthy and/or malignant cells. For these mutations, detection of the wild-type allele is as informative as the presence of the mutant allele . In our dataset, this included a mutation in the X-chromosomal gene, which occurred in cells already carrying loss of chromosome 9, and 3′ UTR mutations in, which also occurred in cells with loss of the other allele on chromosome 9 and chromosome 3.
To add Illumina adapters, dual-indexed barcodes and a custom read primer binding sequence to the fragments, we performed a second PCR using the Streptavidin-bound product as a template , with 2 µl of a 5 µM primer mix and 25 µl PFU Ultra II HS 2× Master Mix .
The enrichment for loci of interest consists of two PCR reactions. For PCR1, to enrich for loci of interest , we designed primers to amplify specific regions . We downloaded the transcript sequence in Geneious Prime 2020, and annotated the mutation of interest. We designed mutation-specific primers within 50 bases upstream of the mutation site . To add a read 2 sequence to this mutation-specific primer, we appended CACCCGAGAATTCCA at the 5′ end.
For PCR2, we used a P5 sequence followed by a 10 bp index barcode and a read 1 sequence as a forward primer and a P7 sequence followed by a 10 bp index barcode and a read 2 sequence as a reverse primer . The PCR was set up as follows: 18 μl of the PCR1 product was added to 2 μl primers and 20 μl Kapa HiFi HotStart ReadyMix.
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