Abstract
Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.
Highlights
Microsatellites are neutral, codominant, highly polymorphic, and abundant genetic markers in eukaryotic genomes [1,2,3]
We considered allelic dropout (ADO) as the failure to amplify one allele in heterozygous individuals [31, 32] and false allele (FA) as an allele-like artifact that is generated by polymerase chain reaction (PCR) [33]
As MHC markers are generally polygenic in vertebrates [38, 39], AmpliSAS might be biased to call a higher number of heterozygotes, yielding a very low rate of dropout, but a very high rate of false alleles
Summary
Microsatellites are neutral, codominant, highly polymorphic, and abundant genetic markers in eukaryotic genomes [1,2,3]. Microsatellites are motifs of one to six base pairs repeated in tandem. High polymorphism in these loci yields high information content from only a few markers [4]. These properties have made microsatellites a widely used tool in very different research areas, such as cancer diagnosis [5], human forensics [6], and conservation biology [7]. During the last twenty years, they have been a powerful and commonly applied tool in molecular ecology for a variety of applications, including individual identification, kinship
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