Highly accurate SNP genotyping from historical and low‐quality samples

Abstract

Historical and other poor-quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error-prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor-quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual-fluorescence genotyping that is very effective for genotyping poorquality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µ µ µ µ L of a 51-bp nuclear sequence. Among samples with ≤ ≤ ≤ ≤ 10 copies/µ µ µ µ L DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.

Back to the future: museum specimens in population genetics

“…SNPs have several advantages, including a known mutation model and a higher genotyping efficiency. Furthermore, they are suitable for highly degraded DNA because genotyping requires only short target DNA sequences (<100 bp) and protocols for genotyping of SNPs have been developed for degraded DNA [59]. However, the discovery of SNPs, that is, the selection of representative samples used for finding polymorphic sites, is crucial and can significantly bias the estimates of genetic variation within and between populations ( [58], and references therein).…”

“…This process can partly be delayed by multiplexing several loci within one PCR reaction, an approach now regularly used in microsatellite and SNP genotyping [59]. PCR multiplex amplification has also been used to obtain the complete sequence of the mitochondrial genome from the woolly mammoth (Mammuthus primigenius) from a 200 mg bone sample [62].…”

Genetic and genomic monitoring with minimally invasive sampling methods

“…A technologically similar platform, Amplifluor SNP genotyping system, has been shown to be highly sensitive with low quality/quantity samples: there was a high level of genotyping success with as few as 10 DNA templates per assay (Morin & Mccarthy, 2007). Mesnick et al, (2011) used 8 microsatellite loci and 38 Amplifluor SNP loci to investigate the population structure of North Pacific sperm whales (Physter macrocephalus).…”

Genome-wide single nucleotide polymorphism (SNP) identification and characterization in a non-model organism, the African buffalo (Syncerus caffer), using next generation sequencing

“…Attention has begun to shift toward SNPs as preferred genetic markers due to their increased power of resolution and accuracy for studying fine scale population structure (Schlötterer, 2004). This is based on their high abundance throughout the genome, simple mutation characteristics, low mutation rates, usability on non-invasive samples and historical DNA, and standardization possibilities between laboratories (Kraus et al, 2014;Morin et al, 2007aMorin et al, ,b, 2004Luikart et al, 2003). SNPs have become an established marker in molecular ecology, evolutionary genetics, and animal breeding (Davey et al, 2011;Kraus et al, 2014Kraus et al, , 2012Morin et al, 2004;Santure et al, 2010).…”