TY - JOUR A1 - Bayerl, Helmut A1 - Kraus, Robert H. S. A1 - Nowak, Carsten A1 - Foerster, Daniel W. A1 - Fickel, Jörns A1 - Kühn, Ralph T1 - Fast and cost-effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next-generation Random Amplicon Sequencing (RAMseq) JF - Molecular ecology resources N2 - 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 (RAMseq), a novel approach for fast and cost-effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs 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). SNPs 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, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies. KW - high-throughput sequencing KW - Lutra lutra KW - nonmodel species KW - RAMseq KW - RAPD KW - variant detection Y1 - 2018 U6 - https://doi.org/10.1111/1755-0998.12717 SN - 1755-098X SN - 1755-0998 VL - 18 IS - 1 SP - 107 EP - 117 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Patel, Riddhi P. A1 - Lenz, Dorina A1 - Kitchener, Andrew C. A1 - Fickel, Jorns A1 - Foerster, Daniel W. A1 - Wilting, Andreas T1 - Threatened but understudied: supporting conservation by understanding the genetic structure of the flat-headed cat JF - Conservation genetics KW - Flat-headed cat KW - Habitat specialist KW - Hybrid capture KW - Mitogenome KW - MtDNA Y1 - 2017 U6 - https://doi.org/10.1007/s10592-017-0990-2 SN - 1566-0621 SN - 1572-9737 VL - 18 SP - 1423 EP - 1433 PB - Springer CY - Dordrecht ER - TY - JOUR A1 - Paijmans, Johanna L. A. A1 - Fickel, Jörns A1 - Courtiol, Alexandre A1 - Hofreiter, Michael A1 - Foerster, Daniel W. T1 - Impact of enrichment conditions on cross-species capture of fresh and degraded DNA JF - Molecular ecology resources N2 - Abstract By combining high-throughput sequencing with target enrichment (‘hybridization capture’), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination-rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter – hybridization temperature – on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect™ arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on-target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments. KW - degraded DNA KW - Felidae KW - hybridization capture KW - mitogenomes KW - next-generation sequencing KW - sequence enrichment Y1 - 2016 U6 - https://doi.org/10.1111/1755-0998.12420 SN - 1755-098X SN - 1755-0998 VL - 16 SP - 42 EP - 55 PB - Wiley-Blackwell CY - Hoboken ER -