DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences

Alquezar-Planas，David E.1,2; Löber，Ulrike1,3,4,8; Cui，Pin1,9; Quedenau，Claudia5; Chen，Wei6,10; Greenwood，Alex D.1,7

2020

10.1111/2041-210X.13497

Methods in Ecology and Evolution   影响因子和分区

2041-210X

2041-210X

There are few available tools to comprehensively and economically identify uncharacterized flanking regions that are not extremely labour intensive and which exploit the advantages of emerging long-read sequencing platforms. We describe SIP; a sonication-based inverse PCR high-throughput sequencing strategy to investigate uncharacterized flanking region sequences, including those flanking mobile DNA. SIP combines unbiased fragmentation by sonication and target enrichment by coupling outward facing PCR priming with long-read sequencing technologies. We demonstrate the effectiveness of SIP by determining retroviral integrations which are high copy and challenging to characterize. We further describe SIP's workflow, examine retroviral (proviral) enrichment and characterize viral structural variants identified. When SIP was coupled with long-read sequencing using the PacBio RS II platform, proviral integration was extensively characterized at high sequence depth per integration. By interrogating the sequence data, we were also able to test several intrinsic factors including SIP's propensity to form chimeric sequences and adapter ligation efficiencies. SIP is an adaption of a traditional molecular biology technique that can be used to characterize any unknown genomic flanking sequence or to extend any sequence for which only minimal sequence information is available. SIP can be applied broadly to study complex biological systems such as mobile genetic elements with high throughput.

inverse PCR KoRV long-read sequencing sonication uncharacterized flanking regions

SCI

英语

其他

Deutscher Akademischer Austauschdienst (DAAD)[2014 57129705] ; Morris Animal Foundation[D14ZO-94]

Environmental Sciences & Ecology

Ecology

WOS:000578968000001

WILEY

2-s2.0-85092611261

Scopus

1.Department of Wildlife Diseases,Leibniz Institute for Zoo and Wildlife Research,Berlin,Germany
2.Australian Museum Research Institute,Australian Museum,Sydney,Australia
3.The Berlin Center for Genomics in Biodiversity Research,Berlin,Germany
4.Experimental and Clinical Research Center,A Cooperation of Charité – Universitätsmedizin Berlin and Max Delbruck Center for Molecular Medicine,Berlin,Germany
5.Genomics,Max Delbrück Center for Molecular Medicine,Berlin,Germany
6.Berlin Institute for Medical Systems Biology,Max-Delbrück Center for Molecular Medicine,Berlin,Germany
7.Department of Veterinary Medicine,Freie Universität Berlin,Berlin,Germany
8.Max Delbrück Center for Molecular Medicine,Host-microbiome factors in cardiovascular disease,Berlin,13125,Germany
9.ShenZhen HaploX Biotechnology Co,LTD,Shenzhen,Nanshan District,518057,China
10.Department of Biology,Southern University of Science and Technology,Shenzhen,518055,China

Alquezar-Planas，David E.,Löber，Ulrike,Cui，Pin,et al. DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences[J]. Methods in Ecology and Evolution,2020,12(1).

Alquezar-Planas，David E.,Löber，Ulrike,Cui，Pin,Quedenau，Claudia,Chen，Wei,&Greenwood，Alex D..(2020).DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences.Methods in Ecology and Evolution,12(1).

Alquezar-Planas，David E.,et al."DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences".Methods in Ecology and Evolution 12.1(2020).