Mseek of Homo sapiens: adult female malignant uterine tumor tissue
The Mseek method of sample processing and deep sequencing of mtDNA, and the procedure for data analysis, were conducted as previously reported with slight modifications (https://doi.org/10.1093/nar/gkv052). Briefly, Mseek consists of (i) digesting linear nuclear DNA (nDNA) with Exonuclease V, (ii) purifying the products using Ampure beads to remove short fragments, (iii) testing the results of the digestion with PCR primers specific for mtDNA and nDNA using one microliter of the digested sample, (iv) Tagmentation, using transposase-based Nextera kit from Illumina, creates fragments, (v) amplifying the library utilizing universal adapters, (vi) size selection using a double-sided bead purification system to select ~350 bp sizes, and (vii) loading samples onto the Illumina NextSeq 500 platform. The sequencing data were generated as fastq files. Briefly, the sequences were filtered for quality (sequences with >10 consecutive nucleotides with Q < 20 were eliminated) and mapped to the revised Cambridge Reference Sequence (rCRS), accession NC_012920. Identical reads were identified as being clonal and were considered only once, irrespective of the number of copies, toward variant calling. A variant call was made only if at least three non-clonal reads were carrying the variant, and minimum coverage of 10 was required at the variant. Variants occurring on reads on one strand (with a skew greater than 0.1 or 10%) of the mtDNA were excluded to reduce errors further. The error rates in NextSeq reads are usually <1 in 1000 (phred score Q > 30), and requiring at least three non-clonal reads reduces the error rate to well under one in a million. Nuclear contamination was estimated using sequences that map to repeat elements such as long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), which only occur in nDNA. This enables reliable estimation of the level of nDNA contamination.