A new approach combining forensic thresholds and a multiple-tubes approach to unravel false microsatellite profiles from cross-contaminated sample material

Abstract

Contamination and degradation are known challenges for reliable genotyping, since they can cause, among other problems, false microsatellite profiles. In this study we described a method to decrease the proportion of false microsatellite profiles from fish scale samples of endangered allis shads (Alosa alosa) from a reintroduction program, where cross-contamination with DNA from other individuals and potentially degradation of samples occurred. To maximize the portion of reliably measurable results, we modified and combined two known approaches—thresholds used in forensic DNA analyses and a multiple-tubes approach. This combined approach increased reliable microsatellite profiles compared with single approaches. The forensic thresholds and the multiple-tubes approach increased the measurable results from 55 to 67% and 75%, respectively, whereas the combined approach accomplished an increase to 90%. This illustrates the potential of the combined approach for other studies with comparable problems or sample material.

How to quantify factors degrading DNA in the environment and predict degradation for effective sampling design

“…We automated this process using the bioinformatics pipeline described in Zarzoso‐Lacoste et al (2018) and applied by Jan et al (2019). This pipeline recovers weaker peaks within a replicate due to hierarchical fall back from more stringent peak detection thresholds, while reducing scoring alleles from cross‐contamination by combining peak height thresholds, peak height ratios, and a multi‐tube approach (Mäck et al, 2021). If more than two alleles were detected at a locus in a replicate, only the highest two were kept, while lower ones, potentially introduced by cross‐contamination, were discarded.…”