Enzymatic removal of Numts from Panthera tigris DNA samples

Forensic mitochondrial DNA analysis is a vital investigative tool for wildlife casework, but numts continue to complicate DNA analysis. A major goal of wildlife forensic DNA analysis is to identify non-human biological evidence to its taxonomic source. Species identification is accomplished by sequencing genetic markers on the mitochondrial genome and comparing evidentiary sequence data to published reference sequences. Due to the high level of sequence similarity between mitochondrial genes and numts, current sequencing methods result in co-amplification of the target gene marker and non-target numt. Co-amplification of target and non-target loci results in ambiguous nucleotide calls in sequence data. Reducing the influence of numts during sequence analysis will provide a technique that will maximize accuracy and minimize error in taxonomic identifications. To overcome the analytical burden of numts, we studied the enzymatic removal of numts using exonuclease V. To evaluate the feasibility of exonuclease V as a numt removal method, total Panthera tigris DNA was extracted from blood and liver and divided into three treatment groups: untreated, 1st digest, and 2nd digest. For the untreated sample, 7.6 % of the 620 bp sequence data was classified as ambiguous. Following treatment, all samples demonstrated a reduction in ambiguous calls: liver-1st digest (48 h): 6.6 %, liver-2nd digest (48 h + 16 h): 1.8 %, and blood-1st digest (48 h): 0 %. Based on this preliminary study, exonuclease V treatment effectively removed numts before sequencing analysis. While exonuclease V treatment has demonstrated potential, additional studies are required to optimize the reaction and fully validate the methodology.