Enrichment of long DNA fragments from mixed samples for Nanopore sequencing

Whole-genome sequencing of pathogenic organisms directly from clinical samples combines 12 detection and genotyping in one step. This can speed up diagnosis, especially for slow-growing 13 organisms like Mycobacterium tuberculosis (Mtb), which need considerable time to grow in 14 subculture, and can...

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Bibliographic Details
Published inbioRxiv
Main Authors Eckert, Sabine E, Chan, Jackie Z-M, Houniet, Darren, The Pathseek Consortium2, Breuer, Judy, Speight, Graham
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 15.04.2016
Subjects
Cell lines
Cytomegalovirus
Deoxyribonucleic acid
DNA
DNA sequencing
Genomes
Genotyping
Influenza A
Nucleotide sequence
Ribonucleic acid
RNA
Subculture
Tuberculosis
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Summary:Whole-genome sequencing of pathogenic organisms directly from clinical samples combines 12 detection and genotyping in one step. This can speed up diagnosis, especially for slow-growing 13 organisms like Mycobacterium tuberculosis (Mtb), which need considerable time to grow in 14 subculture, and can provide vital information for effective personalised treatment. Within the 15 PATHSEEK project, we have developed a bait-capture approach to selectively enrich DNA/RNA from 16 specific bacterial and viral pathogens present in clinical samples. Here, we present a variation of the 17 method that allows enrichment of large fragments of target DNA for sequencing on an Oxford 18 Nanopore MinION sequencer. We enriched and sequenced cDNA from Influenza A (FluA), genomic 19 DNA (gDNA) from human cytomegalovirus (CMV) and from two strains of Mtb, and present an 20 evaluation of the method together with analysis of the sequencing results from a MinION and an 21 Illumina MiSeq sequencer. While unenriched FluA and CMV samples had no reads matching the 22 target organism due to the high background of DNA from host cell lines, enriched samples had 56.7% 23 and 90.9% on-target reads respectively for the best quality Nanopore reads.
DOI:10.1101/048850