TY - JOUR

T1 - Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

AU - Erkes, Annett

AU - Grove, René P.

AU - Žarković, Milena

AU - Krautwurst, Sebastian

AU - Koebnik, Ralf

AU - Morgan, Richard D.

AU - Wilson, Geoffrey G.

AU - Hölzer, Martin

AU - Marz, Manja

AU - Boch, Jens

AU - Grau, Jan

N1 - Funding Information: Open Access funding enabled and organized by Projekt DEAL. This work was supported by grants from the Deutsche Forschungsgemeinschaft ( http://www.dfg.de ) (BO 1496/8-2 to JB, GR 4587/1-2 to JG and FZT 118/2 to SK and MM). Work in RK’s laboratory was supported by a grant from the French Agence Nationale de la Recherche (ANR-2010-BLAN-1723). MM and SK were supported by the Ministry for Economics, Sciences and Digital Society of Thuringia (TMWWDG), under the framework of the Landesprogramm ProDigital (DigLeben-5575/10-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding Information: We acknowledge the financial support of the Open Access Publication Fund of the Martin Luther University Halle-Wittenberg ( https://www.uni-halle.de ).

PY - 2023/12

Y1 - 2023/12

N2 - Background: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

AB - Background: Most plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches. Results: Here, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads. Conclusions: Our results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

KW - Bacteria

KW - Genome assembly

KW - Oxford Nanopore Technologies

KW - Sequencing technologies

KW - Transcription activator-like effectors

UR - http://www.scopus.com/inward/record.url?scp=85150979833&partnerID=8YFLogxK

U2 - 10.1186/s12864-023-09228-1

DO - 10.1186/s12864-023-09228-1

M3 - Article

C2 - 36973643

AN - SCOPUS:85150979833

VL - 24

JO - BMC GENOMICS

JF - BMC GENOMICS

SN - 1471-2164

M1 - 151

ER -