|ZHENG, Z - South China Agricultural University|
|WU, F - South China Agricultural University|
|DENG, X - South China Agricultural University|
Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 5/19/2016
Publication Date: 7/30/2016
Citation: Zheng, Z., Wu, F., Deng, X., Chen, J. 2016. Quick genome sequencing of “Candidatus Liberibacter” strains by use of Enrichment-Enlargement-Next generation sequencing (EEN). American Phytopathological Society Abstracts. 106:S4.26.
Technical Abstract: Members of “Candidatus Liberibacter” are associated with several important plant diseases such as citrus Huanglongbing (HLB) and potato zebra chip (ZC) disease. Inability to culture and low titers in infected hosts have been major obstacles for research on these bacteria. The use of whole genome sequence analysis could be an effective means to characterize and study the bacteria. Next Generation Sequencing (NGS) technology with enrichment-enlargement-NGS (EEN) was used for quick genome sequencing of liberibacters. Enrichment includes increasing the ratio of liberibacter DNA to host DNA through using alternative hosts with higher bacterial titer, removal of the highly methylated plant DNA, and selection of samples with low Ct values for liberibacter DNA. Enlargement includes increasing enriched DNA quantity through Multiple Displacement Amplification (MDA) to meet requirements of a sequencing platform. The NGS step includes selection of a sequencing platform and development of specific pipelines/procedures for liberibacters. With EEN, >10 G of sequence data were generated from MiSeq, followed by collection of liberibacter reads using Perl scripts, de novo and/or 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 either plant or psyllid hosts. The EEN procedure has facilitated research on HLB liberibacters recently found in California and could be applied to genome research in other fastidious prokaryotes.