Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

Abstract

The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3= end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.T he use of high-throughput sequencing technologies (1, 2) has transformed the field of microbial ecology by contributing to a significant body of work that has changed our understanding of microbially diverse populations in a range of ecosystems. This is particularly true for investigations of bacterial and archaeal communities that target the 16S rRNA gene (1, 3-5). However, amplicon sequencing approaches for eukaryotes hav...

Planktonic foraminifera-derived environmental DNA extracted from abyssal sediments preserves patterns of plankton macroecology

“…The analysis based on relative abundances yields a pattern with highly consistent results for comparisons between climatic zones and more scatter when comparing samples within a region or within one climatic zone. This is likely due to the fact that the eDNA data only cover a part of the morphological diversity of the foraminifera combined with differential distortion of the original abundance signal due to variation in gene copy number (Weber and Pawlowski, 2013) and primer bias (Bradley et al, 2016).…”

Validation of a universal set of primers to study animal‐associated microeukaryotic communities

“…The bias is an issue with all methods, and generally samples analysed in the same way give a relative picture, while samples treated in different ways are hard to compare, and this method is much the same in that regard (Hadziavdic et al, 2014). However, this is only a limitation because the size of the amplicon generated by the UNonMet-PCR is slightly larger than can be handled by current Illumina technologies (Bradley et al, 2016). It is not unreasonable to assume that these limitations will soon be eliminated by changes in the sequencing platforms and chemistry that will allow the UNonMet-PCR to be used directly for Illumina libraries (or other chemistries) (van Dijk et al, 2014), which would also make the process even simpler and less expensive.…”

Bacteria and microeukaryotes are differentially segregated in sympatric wastewater microhabitats

“…Second, variation in the 18S rRNA gene copy number can be large between microeukaryotic organisms (Prokopowich et al . , Bradley et al ., ). Thus, the ecological interpretation of the microeukaryotic community based on sequence abundance should be taken with appropriate caution.…”

“…Applying primers specific for distinct protist groups may allow obtaining a higher resolution (Lara et al, 2007;Glaser et al, 2014) and retrieving sequences of particular groups of interest which would be missed by universal primers even in a high-throughput sequencing approach (Lentendu et al, 2014). Second, variation in the 18S rRNA gene copy number can be large between microeukaryotic organisms (Prokopowich et al 2003, Bradley et al, 2016. Thus, the ecological interpretation of the microeukaryotic community based on sequence abundance should be taken with appropriate caution.…”