The effect of metabarcoding 18S rRNA region choice on diversity of microeukaryotes including phytoplankton

A comparison of two gene regions for assessing community composition of eukaryotic marine microalgae from coastal ecosystems

“…The V9 region generated more raw reads than the V4 at both sites. This aligns with other studies assessing both the EC 42 and EMC, where up to twice as many raw reads were produced by the V9 30 , 42 . Greater raw reads generated when using V9 are most likely a product of the shorter fragment length for this gene region.…”

“…It is widely acknowledged that the number of sequences for V4 and V9 regions differ across open-source sequence databases [Refs. 30 , 46 ]. Primer bias could also contribute to this, though interestingly, the V4 was identified by Hadziavdic, Lekang 53 along with the 18S V2 and V9 as being best suited for biodiversity assessments due to the quality of universal primers.…”

“…It also removes the necessity for databases to fully resolve taxa to species level, which is one of the main limitations of metabarcoding when used to characterise EMCs. Gene region and primer selection can also bias the diversity observed in communities when using metabarcoding approaches and requires careful consideration 30 – 32 .

Figure 1 The high-level taxonomic groups of eukaryotic marine microalgae used in this study.

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“…Comparisons of the 18S rDNA V4 and V9 regions, and their coverage of diversity within EMCs has been assessed 30 – 32 . However, the focus is usually on the depth of taxonomic resolution and total diversity observed.…”

A full‐length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing

“…The 18S rDNA gene is one of the most commonly used markers for species identification and delineation due to its conserved and hypervariable regions (V1‐V9) (Kooistra et al., 2010 ; Medlin et al., 1996 ). Previous studies of protist diversity using metabarcoding have used one of the hypervariable short read regions in the 18S rDNA e.g., V1‐V2 and V3, V4, V6, V7‐V8, V8‐V9 or V9 (Amaral‐Zettler et al., 2009 ; Bradley et al., 2016 ; Bukin et al., 2023 ; de Vargas et al., 2015 ; Hadziavdic et al., 2014 ; Hatteranth‐Lehmann et al., 2019 ; Massana et al., 2015 ; Medinger et al., 2010 ; Mohrbeck et al., 2015 ; Piredda et al., 2017 ; Stoeck et al., 2010 ). Until now, most metabarcoding studies of plankton diversity has been assessed using primer pairs that produce fragments lengths <600 bp.…”