Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

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.