Eckert, SE;
Chan, JZ-M;
Houniet, D;
PATHSEEK consortium, .;
Breuer, J;
Speight, G;
(2016)
Enrichment by hybridisation of long DNA fragments for Nanopore sequencing.
Microbial Genomics
, 2
(9)
, Article e000087. 10.1099/mgen.0.000087.
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Abstract
Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short fragments of around 200-300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait-capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenza virus A, human cytomegalovirus (HCMV) and genomic DNA from two strains ofMycobacterium tuberculosis(M. tb) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION™ and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION™ platform had 57 % and 99 % best-quality on-target reads respectively.
| Type: | Article |
|---|---|
| Title: | Enrichment by hybridisation of long DNA fragments for Nanopore sequencing |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1099/mgen.0.000087 |
| Publisher version: | https://doi.org/10.1099/mgen.0.000087 |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Mycobacterium tuberculosis, enrichment by hybridisation, human Herpes virus/cytomegalovirus, influenza A, nanopore sequencing |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Genetics and Genomic Medicine Dept UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10044300 |
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