Poster: Biology & Disease Mgmt: Bacteriology
Quick genome sequencing of “Candidatus Liberibacter” strains by use of Enrichment-Enlargement-Next generation sequencing (EEN)
J. CHEN (1), Z. Zheng (2), F. Wu (3), X. Deng (3) (1) USDA-ARS, U.S.A.; (2) South China Agricultural University, China; (3) South China Agricultural University, China
Members of “Candidatus Liberibacter” are associated with several important plant diseases such as citrus Huanglongbing (HLB) and potato zebra chip (ZC) disease. The inability to culture and the low titers in infected hosts of the bacteria have been major obstacles for research. Whole genome sequence analysis could be an effective means to study the bacteria. Taking the advancement of Next Generation Sequencing (NGS) technology, a procedure, called EEN (enrichment-enlargement-NGS), has been developed for quick genome sequencing of liberibacters. Enrichment includes increasing the DNA ratio of liberibacter to its host using alternative hosts with higher titer capacity, removal of the highly methylated plant DNA and selection of low Ct values samples; Enlargement includes increasing the enriched DNA quantity through whole DNA amplification to meet the requirement of a sequencing platform; And the NGS step includes selection of sequencing platform and development of liberibacter-specific pipelines. With EEN, >10 G of sequence data was generated from MiSeq, followed by collection of liberibacter reads using Perl scripts, de novo or/and referenced genome assembling, and sequence annotation. Thus far, seven draft genome sequences have been generated, five HLB liberibacters (China, California, and Florida) and two ZC liberibacters (California) from plant or psyllid hosts. The EEN procedure could also benefit genome research in other fastidious prokaryotes.