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Fast and cost‐effective single nucleotide polymorphism ( SNP ) detection in the absence of a reference genome using semideep next‐generation Random Amplicon Sequencing ( RAM seq)
Author(s) -
Bayerl Helmut,
Kraus Robert H. S.,
Nowak Carsten,
Foerster Daniel W.,
Fickel Joerns,
Kuehn Ralph
Publication year - 2018
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/1755-0998.12717
Subject(s) - biology , genotyping , amplicon , genetics , snp genotyping , single nucleotide polymorphism , molecular inversion probe , microsatellite , snp , genetic diversity , population , genotype , allele , polymerase chain reaction , gene , demography , sociology
Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing ( RAM seq), a novel approach for fast and cost‐effective detection of single nucleotide polymorphisms ( SNP s) in nonmodel species by semideep sequencing of random amplicons. By applying RAM seq to the Eurasian otter ( Lutra lutra ), we identified 238 putative SNP s after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction ( PCR ) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNP s produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAM seq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.