Allelic mismatch
Pairwise comparisons of technical replicates in the contig dataset showed that SNP calls were on average 18.3% (SD = 6.0) different between technical replicates (Table 2; Fig. 3A). When looking at only homozygous call differences, however, this value fell to 0.13% (SD = 0.1; Fig. 3B), suggesting that almost all allelic mismatches were driven by differences in whether a given SNP was called as homozygous or heterozygous. Supernatant and pellet replicates had the most shared SNP calls among capture-based replicates, with heterozygous differences of 11.9% (SD = 5.2) of SNPs on average, while formalin-fixed replicates differed 22.2% (SD = 4.7) from supernatant replicates and 18.0% (SD = 4.4) from pellet replicates. Capture-based replicates exhibited fairly consistent rates of mismatch to the RADseq replicates with mean differences of 20.1% (SD = 1.6), 22.9% (SD = 3.4), and 20.4% (SD = 1.1) in heterozygous/homozygous calls for supernatant, formalin-fixed, and pellet replicates respectively. This difference dropped to < 0.26% for non-heterozygous/homozygous differences. If differences in heterozygous/homozygous calls are random we would expect a roughly 50/50 balance of heterozygous versus homozygous calls between replicates. However, we identified a systematic imbalance between heterozygous and homozygous calls, where >85% (SD = 4.98) of differences between capture and RADseq replicates were explained by a heterozygous call in the target capture replicate and a homozygous call in the RADseq replicate (Fig. S2). This consistent and significant imbalance was not observed among the capture-based technical replicates although formalin-fixed replicates exhibited greater deviation and variance from 50/50 balance than other replicate types (Fig. S2).
Table 2: Results of allelic mismatch analyses. N = number of samples, Sites = the number of SNPs shared on average between technical replicates in each comparison. Het = the average percentage of sites that exhibit different heterozygous/homozygous calls for each comparison, Hom = the percentage of sites that exhibit a homozygous call difference on average for each comparison, Allele balance = the average percentage of heterozygous/homozygous differences that are heterozygous in the first replicate listed. RADseq comparisons are bolded to highlight differences.