Data from: Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus

The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecological studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each specimen studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences. With this approach, we show that: i) contaminants accounted for a negligible proportion of the bacteria identified in our samples; ii) the taxonomic composition of our samples and the relative abundance of reads assigned to a taxon were very similar across PCR and DNA replicates for each aphid sample; in particular, bacterial DNA concentration had no impact on the results. Furthermore, by analysing the distribution of unique sequences across samples rather than aggregating them into operational taxonomic units (OTUs), we gained insight into the specificity of endosymbionts for their hosts. Our results confirm that Serratia symbiotica is often present in Cinara species, in addition to the primary symbiont, Buchnera aphidicola. Furthermore, our findings reveal new symbiotic associations with Erwinia and Sodalis-related bacteria. We conclude with suggestions for generating and analysing 16S rRNA gene sequences for arthropod endosymbiont studies.

栖息于节肢动物体内的细菌群落，通常以少数几种内共生菌为优势类群，这些内共生菌对宿主的生态功能具有重要作用。相较于跨样本比较细菌物种丰富度，节肢动物内共生菌的生态学研究往往更侧重于鉴定每份研究标本所关联的主要细菌菌株。因此，从分析结果中剔除污染物并对序列开展精准的分类学注释，是节肢动物微生物组研究中的关键环节。本研究旨在验证一套用于探究蚜虫相关内共生细菌的Illumina 16S rRNA基因测序实验方案与分析流程。我们利用12个物种（蚜科：长管蚜亚科，Cinara属）的重复DNA样本及多组对照，剔除了每个样本中读段数未达到最低阈值的单独序列，并对剩余序列开展分类学注释。通过该方法，我们得到如下结果：(i) 污染物在本研究样本中鉴定出的细菌类群里占比极低；(ii) 每份蚜虫样本的PCR重复与DNA重复之间，样本的分类学组成以及分配至某一分类单元的读段相对丰度均高度相似；尤其值得注意的是，细菌DNA浓度对实验结果并无影响。此外，相较于将独特序列聚类为操作分类单元（operational taxonomic units, OTUs），我们通过分析独特序列在各样本中的分布情况，得以深入探究内共生菌对宿主的特异性。我们的研究结果证实，Cinara属物种中除初级共生菌蚜虫布赫纳共生菌（Buchnera aphidicola）外，通常还存在共生沙雷氏菌（Serratia symbiotica）。此外，我们的研究还发现了与欧文氏菌属（Erwinia）和索达尔氏菌属（Sodalis）相关细菌的新型共生关联。最后，我们针对节肢动物内共生菌研究中的16S rRNA基因序列获取与分析工作提出了相关建议。