SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

Weili Cai,Michael J Stulberg,Schyler Nunziata,John Rascoe

doi:10.1038/s41598-019-55144-4

Abstract

Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Highlights

Huanglongbing (HLB), or citrus greening, is a devastating citrus disease caused by phloem-restricted gram-negative bacteria ‘Candidatus Liberibacter’ spp[1,2]
The released Candidatus Liberibacter asiaticus’ (CLas) genomes were obtained from either highly infected psyllids or citrus samples[14,15,16,17] because the whole genome sequence of CLas can only be obtained using metagenomic sequencing, due to the lack of in vitro culture. Such high pathogen titer samples are needed because a low percentage of sequencing reads belonging to CLas are present in a metagenomic sample, primarily because of large genome size difference between pathogen and host and relative low copy number of pathogen DNA
Pathogen DNA is enriched from 500- to 45,000-fold compared to non-enriched samples. All these results suggest that Agilent SureSelect XT HS target enrichment can effectively capture target DNA from complex CLas samples and significantly increase the pathogen DNA ratio

Summary

Introduction

Huanglongbing (HLB), or citrus greening, is a devastating citrus disease caused by phloem-restricted gram-negative bacteria ‘Candidatus Liberibacter’ spp[1,2]. The released CLas genomes were obtained from either highly infected psyllids or citrus samples (equivalent to 18 to 23 Cq using Li 16S qPCR)[14,15,16,17] because the whole genome sequence of CLas can only be obtained using metagenomic sequencing, due to the lack of in vitro culture. The probe-bound DNA is eluted and collected for further NGS application, and often has much higher target DNA concentration than the original input samples[19,20] This method has been widely used to capture and enrich targeted DNA from complex biological samples, but is not commonly used to recover plant pathogens from a plant host background[21,22,23]

Methods

Results

Conclusion