Comparing metatranscriptomics, PCR-based methods, and morphology in estimating diversity and composition of zooplankton mock communities

Studying complex metazoan communities requires taxonomic expertise and laborious work if done using the traditional morphological approach. Nowadays, popular use of molecular25 based methods accompanied by massively parallel sequencing (MPS) provides rapid higher resolution diversity analyses. However, estimates derived from molecular-based approach can be biased co-detection environmental DNA (eDNA), pseudogene contamination, PCR amplification biases. Here, we constructed microcrustacean zooplankton mock to compare species composition PCR-based genomic (gDNA) complementary (cDNA), metatranscriptomic transcripts, morphology data. Mock community analyses show that gDNA mitochondrial cytochrome c oxidase I (mtCOI) amplicons inflate richness due nontarget sequence contamination. Significantly amplicon variant (ASV) nucleotide in than cDNA indicated presence putative pseudogenes. Last, failed detect most abundant priming site mismatch. Overall, transcripts provided closely resembled The was further tested field samples. results showed it could provide consistent among biological technical replicates while allowing monitoring temporal changes different markers. These findings characterization on reflects actual more approaches.