title: A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits.
abstract: The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.
authors.affiliation: Department of Zoology, University of Oxford, Oxford OX1 3PS, UK timokratis.karamitros@zoo.ox.ac.uk.
authors.forename: Timokratis
authors.initials: T
authors.lastname: Karamitros
authors.affiliation: Department of Zoology, University of Oxford, Oxford OX1 3PS, UK timokratis.karamitros@zoo.ox.ac.uk.
authors.forename: Gkikas
authors.initials: G
authors.lastname: Magiorkinis
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D020459:Genes, rRNA; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009876:Operon; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*