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A high-throughput multiplexing and selection strategy to complete bacterial genomes.

Sergio Arredondo-Alonso1,2, Anna K Pöntinen1, François Cléon3

  • 1Department of Biostatistics, University of Oslo, 0317, Oslo, Norway.

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Oxford Nanopore Technologies' 96-barcode kit enables high-throughput bacterial genome sequencing. This multiplexing approach, combined with smart isolate selection, efficiently generates complete bacterial genomes and resolves plasmid sequences.

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Area of Science:

  • Genomics
  • Microbiology
  • Bioinformatics

Background:

  • Short-read sequencing yields draft bacterial genomes.
  • Long-read sequencing can complete genomes but is costly.
  • High-throughput sequencing of bacterial isolates is limited by cost.

Purpose of the Study:

  • Evaluate Oxford Nanopore Technologies (ONT) 96-barcode kit for high-throughput complete bacterial genome generation.
  • Propose an isolate selection strategy for optimizing long-read sequencing in large bacterial collections.

Main Methods:

  • Utilized ONT's 96-barcode kit for multiplexed long-read sequencing of 96 Escherichia coli isolates.
  • Generated both hybrid (short-read + long-read) and ONT-only assemblies.
  • Developed and applied an isolate selection strategy for representative sampling.

Main Results:

  • Achieved near-complete chromosomal sequences (contiguity=0.89) and high plasmid replicon contiguity (0.98) despite uneven read distribution.
  • Demonstrated the suitability of multiplexing for resolving plasmid sequences.
  • Showcased the value of combining ONT and short-read data for unbiased isolate selection, optimal genome accuracy, and inclusion of small plasmids.

Conclusions:

  • The proposed long-read isolate selection strategy ensures bacterial genome completion across diverse collections.
  • The multiplexing approach holds potential for high-throughput bacterial genome closure.
  • Complete bacterial genome sequencing is feasible on a large scale using this method.