Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error

Abstract

Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: , and We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size.

Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single‐cell high‐throughput DNA sequencing

“…Gong et al () and Wang et al () showed that the minimal similarity between two copies of SSU rRNA gene in a single ciliate cell was more than 99% and confirmed the reliability of the commonly used OTU clustering cutoff of 95–99% for ciliates. The data of the previous study of Gong et al () and Wang et al () were based on a clone library and Sanger sequencing approach, while only < 50 sequences were sampled from each cell. Rarefaction analysis of ciliate haplotypes, therefore, indicated the presence of a higher number of haplotypes in a single cell than what was sampled (Gong et al ).…”

Genome analyses of the new model protist Euplotes vannus focusing on genome rearrangement and resistance to environmental stressors

“…One of the most diverse and highly differentiated group among single‐celled microorganisms are ciliates, which emerged approximately one billion years ago (Parfrey, Lahr, Knoll, & Katz, ) and are abundant in diverse habitats across the globe, where they are among the most important components of food webs in aquatic ecosystems (Gao et al, ; Lynn, ). Ciliate diversity, physiology and abundance have been linked to studies of environmental change (Gong, Song, & Warren, ; Xu, Zhang, Jiang, & Yang, ), pollution monitoring (Gutiérrez, Martín‐González, Díaz, & Ortega, ; Jiang et al, ; Stoeck, Kochems, Forster, Lejzerowicz, & Pawlowski, ), biogeography (Foissner, Chao, & Katz, ; Liu et al, ; Petz, Valbonesi, Schiftner, Quesada, & Cynan Ellis‐Evans, ), adaptive evolution (Clark & Peck, ; He et al, ), cell biology (Jiang, Zhang, Vallesi, Yang, & Gao, ; Wang, Zhang, et al, ; Zheng et al, ) and epigenetics (Wang, Chen, Sheng, Liu, & Gao, ; Xiong et al, ; Zhao, Wang, Wang, Liu, & Gao, ).…”

GPSit: An automated method for evolutionary analysis of nonculturable ciliated microeukaryotes

“…Subsequently, using sequence information from multiple gene markers (18S‐ITS‐28S rDNA, alpha‐tubulin gene, COI, etc.) has become the widely‐accepted principle in phylogenetic analysis, especially for ciliates, an ecologically important protist group with a high degree of morphological complexity (Chen et al., ; Fernandes, da Silva Paiva, da Silva‐Neto, Schlegel, & Schrago, ; Gao, Gao, Wang, Katz, & Song, ; Gao & Katz, ; Gao, Song, & Katz, ; Gao, Li et al., ; Gao, Warren et al., ; Gao et al., ; Huang, Luo, Bourland, Gao, & Gao, ; Wang, Zhang et al., ; Wang, Wang et al., ; Wang, Wang, Sheng et al., ; Wang, Sheng et al., ; Yan et al., ; Yi, Huang, Yang, Lin, & Song, ; Zhang et al., ; Zhao et al., ).…”