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Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example
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Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing.

Annett Erkes1, René P Grove2, Milena Žarković3

  • 1Institute of Computer Science, Martin Luther University Halle-Wittenberg, 06120, Halle, Germany.

BMC Genomics
|March 27, 2023
PubMed
Summary
This summary is machine-generated.

Oxford Nanopore Technologies (ONT) sequencing combined with a computational correction pipeline successfully assembled Xanthomonas genomes and their complete transcription activator-like effector (TALE) repertoires (TALomes). This method offers an accurate and efficient approach for large-scale bacterial genomics.

Keywords:
BacteriaGenome assemblyOxford Nanopore TechnologiesSequencing technologiesTranscription activator-like effectors

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Area of Science:

  • Genomics
  • Microbiology
  • Bioinformatics

Background:

  • Plant-pathogenic Xanthomonas bacteria utilize transcription activator-like effectors (TALEs) to manipulate host genes and facilitate infection.
  • The complete set of TALE genes in a Xanthomonas strain is termed the TALome, often found in gene clusters that challenge genome assembly.
  • Standard sequencing methods struggle to fully resolve TALE-carrying Xanthomonas genomes due to conserved repeat structures.

Purpose of the Study:

  • To successfully assemble the genomes of five Xanthomonas strains using Oxford Nanopore Technologies (ONT) sequencing data.
  • To develop and validate a computational correction pipeline for accurately reconstructing TALomes from ONT-assembled genomes.
  • To demonstrate the efficiency and capacity of multiplexed ONT sequencing for large-scale TALome characterization.

Main Methods:

  • Genome assembly of five Xanthomonas strains utilizing Oxford Nanopore Technologies (ONT) long-read sequencing data.
  • Development of a specialized computational pipeline to correct errors in TALE genes within ONT assemblies, particularly homopolymer regions.
  • Assessment of the multiplexing capacity of ONT sequencing for simultaneous analysis of multiple samples.

Main Results:

  • Achieved successful assembly of the 5 Mbp genomes for five Xanthomonas strains using ONT sequencing.
  • Demonstrated that ONT reads are superior to Illumina and PacBio reads for resolving complex genomes like Xanthomonas oryzae pv. oryzae (Xoo) PXO35.
  • The computational correction pipeline yielded TALome accuracy comparable to Illumina-based polishing, effectively resolving TALE gene sequences from ONT data.
  • Reconstructed the complete TALome of Xoo PXO35 from fewer than 20,000 ONT reads when combined with computational correction.

Conclusions:

  • Multiplexed ONT sequencing coupled with a TALE-gene-specific computational correction pipeline is a powerful tool for future TALome characterization.
  • This approach enables high-throughput analysis of TALomes in large collections of Xanthomonas strains.
  • The study highlights the potential of ONT sequencing for resolving complex bacterial genomes and gene families crucial for plant-pathogen interactions.