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| Item Type: | Article |
|---|---|
| Title: | DNA sonication inverse PCR for genome scale analysis of uncharacterized flanking sequences |
| Creators Name: | Alquezar‐Planas, D.E. and Löber, U. and Cui, P. and Quedenau, C. and Chen, W. and Greenwood, A.D. |
| Abstract: | 1. 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. 2. 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. 3. 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. 4. 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. |
| Keywords: | Inverse PCR, KoRV, Long‐Read Sequencing, Sonication, Uncharacterized Flanking Regions |
| Source: | Methods in Ecology and Evolution |
| ISSN: | 2041-210X |
| Publisher: | Wiley |
| Date: | 22 September 2020 |
| Official Publication: | https://doi.org/10.1111/2041-210X.13497 |
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