Amplicon library labelled TPH06 from PCR amplified genetic barcodes from tumours, metastases and needles samples.
PCR was performed on crude tumor lysates diluted 1:10 in water. Forty microlitres of lyseate was mixed with PCR master mix in 96-well plates and split into replicate reactions to assess barcode detection reliability. cfDNA and lung samples were run in quintuplicate. Barcodes were amplified using primers TopLib (5-TGCTGCCGTCAACTAGAACA-3) and BotLib (5-GATCTCGAATCAGGCGCTTA-3) under standard cycling conditions, followed by a second PCR to add Illumina indexes. Amplicons (266 bp) were verified by agarose gel electrophoresis, pooled, bead-purified, and sequenced on an Illumina NextSeq. Sequencing parameters: No index reads, spike in 25% PhiX