TY  - JOUR
A1  - Cui, Pin
A1  - Löber, Ulrike
A1  - Alquezar-Planas, David E.
A1  - Ishida, Yasuko
A1  - Courtiol, Alexandre
A1  - Timms, Peter
A1  - Johnson, Rebecca N.
A1  - Lenz, Dorina
A1  - Helgen, Kristofer M.
A1  - Roca, Alfred L.
A1  - Hartman, Stefanie
A1  - Greenwood, Alex D.
T1  - Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome
JF  - PeerJ
N2  - Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small.
KW  - Integration sites
KW  - Retroviral endogenization
KW  - KoRV
KW  - Target enrichment
KW  - Clustering
Y1  - 2016
U6  - https://doi.org/10.7717/peerj.1847
SN  - 2167-8359
VL  - 4
PB  - PeerJ Inc.
CY  - London
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  -