Data from: A comparison between transcriptome sequencing and 16S metagenomics for detection of bacterial pathogens in wildlife

Background: Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings: We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance: We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence.

研究背景：啮齿动物是引发人类与畜禽众多人畜共患病的病原体的主要储存宿主。在个体与种群层面评估其微生物多样性，对于监测地方性感染、揭示储存宿主内的微生物关联模式至关重要。近年来，下一代测序（Next-Generation Sequencing，NGS）技术已被用于解析不同生态系统的微生物群落，但尚未有研究对不同NGS方法的相对效能进行评估。本研究对比了两种NGS技术——RNA测序（RNA-Sequencing，RNA-Seq）与16S宏基因组学（16S-metagenomics），以评估二者在啮齿动物种群中筛查被忽视的人畜共患细菌的能力。

研究方法与主要结果：我们首先从法国采集的190只田鼠的脾脏中提取核酸。将RNA提取物混合后进行随机反转录，随后利用HiSeq平台开展RNA测序。将组装得到的细菌序列比对至GenBank收录的最相近分类单元。采用两种测序平台——454 GS-FLX与MiSeq，通过16S宏基因组学方法对DNA提取物进行分析。使用带条形码的通用引物对每个样本的16S rRNA编码基因的V4区域进行扩增。将扩增产物进行多重化处理后，在不同测序平台上完成测序。对得到的测序数据进行去多重化处理，随后通过分析流程，利用核糖体数据库项目（Ribosomal Database Project，RDP）对每一条读段进行分类学注释。
共计检测到45个致病细菌属。RNA测序鉴定得到的细菌种类与采用MiSeq平台的16S宏基因组学方法（16S-MiSeq）检测结果相当。与之相反，采用454焦磷酸测序的16S宏基因组学方法（16S-454）未能检测到其中21种病原体。此外，16S宏基因组学方法揭示了棕背田鼠体内存在高水平的混合感染。

结论与意义：本研究认为，RNA测序与16S-MiSeq方法在检测细菌时具有同等的灵敏度。尽管仅16S-MiSeq方法可实现对单个储存宿主体内细菌的鉴定，进而推导宿主种群中细菌的感染率，并构建储存宿主内的细菌相互作用模式。此外，通过16S-MiSeq方法得到的细菌读段数可作为细菌感染率的良好替代指标。