Extended Data Fig. 1: Minimal host DNA is found in cancer hemolymph samples.
(a) Hemolymph images for the four clams in this study sampled 2018–21. The other seven clams sampled 2010–14 were reported in past studies by Arriagada & Metzger et al. (2014) and Metzger et al (2015). Scale bars are 50 µm. Fraction of cancer cells detected by MarBTN-specific qPCR, as reported by Giersch et al. (2022), are included in the lower left of each image. Note that while this assay is highly sensitive for the detection of low levels of MarBTN infection in animals, the fraction is a ratio of two qPCR values and minor variation in qPCR values can lead to large variation in the fraction when it is close to 100% cancer. (b) We identified SNVs in mitochondrial DNA in each individual sample and used the median VAF of those SNVs to estimate the purity of the sample. Number of loci: 21, 20 and 13 for healthy clams as ordered in figure, 53 (PEI) and 46 (USA) likely somatic for MarBTN samples. (c) Since mitochondrial genome copy numbers may differ between host and MarBTN cells, we also identified homozygous nuclear SNVs in regions called as copy number 2 in both sub-lineages and used the median VAF of those SNVs to estimate the purity of the sample (number of loci: 250,000 for non-reference healthy clams, 15,000 MarBTN-specific loci for MarBTN samples). Values for pure samples would be expected to be slightly below one due to mapping/sequencing errors, as evidenced by the healthy clams, which serve as pure sample controls (black, all DNA is from one individual). In cancer samples, deviation below this near-one value is attributed to the presence of contaminating host DNA (DNA is a mixture of two individuals – the cancer and the host). Two MarBTN isolates that were excluded from this study due to high host DNA contamination are included on this plot as contaminated sample controls (gray). Both nuclear and mitochondrial markers calculations yield similar estimates of cancer cell purity 96% or greater. MtDNA has the advantage of all loci being ‘homozygous’ and much greater depth than nuclear, giving more resolution as to the exact cancer cell percentage. However, mtDNA copies per cell may vary from sample to sample and between host and cancer. We also extracted DNA from tissue samples for a subset of the USA cancers and estimated the fraction of cancer DNA disseminated into tissue using the same methodology for mitochondrial (d) and nuclear (e) loci. Tissue samples contain variable and in some cases quite high, fractions of cancer DNA. This made genome-wide differentiation between host and cancer SNVs difficult in tissue and lead us to not include paired tissue DNA in our analyses, instead relying on variant calling thresholds to eliminate host variants from our cancer variant calling pipelines. Box plots display ggplot defaults - median (center), interquartile range (box), and the less extreme of minima/maxima or 1.5* interquartile range (whiskers).
