Fresh is best: Accurate SNP genotyping from koala scats

Abstract

Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next-generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq^™ protocol. We compare genotype results from two-day-old scat DNA and 14-day-old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14-day-old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq^™ process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.

Keywords: SNP Genotyping; diet; disease; koala; scat.

Figure 1

Two‐day‐old versus 14‐day‐old scat DNA (a) missing data and (b) genotyping error, when compared to template genotype from blood. Missing data (a) shows percentage of loci (n = 1272) which do not provide any read data in scat samples. Read error and null allele (b) show percentage of remaining loci which do not match blood template genotype due to incorrect read or allelic dropout. Sample size: two‐day‐old samples: n = 10; 14‐day‐old samples: n = 7

Figure 2

Distribution of reference allele sequencing depths of 1272 SNP loci for blood DNA extractions, two‐day‐old scat DNA extractions, and 14‐day‐old scat DNA extractions. Average sequence depth across all loci: Blood: Ref allele—49X, SNP allele—31X; Two‐day‐old scat: Ref allele—4.3X, SNP allele—3.2X; 14‐day‐old scat: Ref allele—6.1X, SNP allele—3.8X

Figure 3

Neighbor‐joining tree of genetic distances using 209 highly conserved SNP loci for blood and scat DNA samples. Loci selected for genetic distance calculation was based on sorting for sequencing depth, error rates, and homozygous loci. Scat DNA samples with missing information at more than 50% of loci were excluded from this analysis