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

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 16S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

黄龙病（HLB）是一种全球性的致命柑橘病，由亚洲柑橘木虱传播的质体限制性细菌‘Candidatus Liberibacter asiaticus’（CLas）引起。为了有效管理这种疾病，理解不同地理位置分离的细菌之间的关系至关重要。全基因组测序方法将为不同种群间的多样性提供更精确的分子特征描述。由于缺乏体外培养，获取CLas的全基因组序列仍然是一个挑战，尤其是在中等至低滴度样本中。为了从HLB阳性柑橘样本中获得良好的CLas覆盖，需要数亿个测序读数。为了克服这一限制，我们在此提出一种新的方法，即Agilent SureSelect XT HS目标富集技术，该技术能够从宏基因组样本中特异性地富集CLas，同时大幅降低成本并提高病原体全基因组覆盖度。在本研究中，成功对CLas基因组进行了测序，基因组覆盖率达到99.3%，测序覆盖度超过72倍，来自低滴度组织样本（相当于使用Li 16S qPCR测定的28.52 Cq）。更重要的是，该方法还能有效捕捉CLas基因组中的多样性区域，为不同菌株提供精确的分子特征描述。