Environmental DNA (eDNA) has acquired importance over the last decade growing quickly along with laboratory and sequencing techniques. Despite eDNA analysis is a cost-effective non-invasive method for monitoring biodiversity, there's still a need to investigate factors affecting final outputs. We explored protocols for each step of the eDNA analysis, and mock communities were created to delve deeper into the variables influencing results. 13 DNA fish extracts were used to compose non-normalized (MC1) and normalized (MC2) mock communities. To investigate the mitochondrial DNA (mtDNA) proportion in fish extracts, MC1 and MC2 were created with pre-PCR extracts (before the 1st step PCR of the library preparation) and amplified extracts (post-PCR) by using tele02 primers targeting the 12S ribosomal RNA (rRNA) gene. The DNA library was prepared with a two-step approach, samples were pooled after indexing and sequenced on MiSeq Illumina (2X250PE). Demultiplexed sequences were processed with the ObiTools pipeline launched in Galaxy.eu. The parameters were set based on known species composition. We reported a significant and positive correlation (Pearson=0.9, p 0.05) between the number of reads and concentration only when the target gene is available in a high number of copies (MC1 post-PCR). Additionally, as expected, the Relative Read Abundance (RRA) pointed out an even proportion among species in MC2 (E=0.91) and MC1 (E=0.90) post-PCR, in contrast with a lack of evenness in MC2 (E=0.68) and MC1 (E=0.49) pre-PCR. We also highlighted that primer efficiency may vary among fish extracts and depending on starting concentrations, as no reads were assigned to Mullus barbatus in MC1 pre-PCR regardless of the starting concentration (13 ng/μl). Conversely, M. barbatus showed 10% RRA in MC1 post-PCR (averaged RRA/species was 7.86%). These results suggest that in real communities some species are less likely to be detected due to mtDNA starting concentration and primer efficiency.
Validation of a pipeline for eDNA analysis by mock communities: evaluation of variables influencing mitochondrial DNA abundance
Giulia Mariani;Ludovica Di Renzo;
2024-01-01
Abstract
Environmental DNA (eDNA) has acquired importance over the last decade growing quickly along with laboratory and sequencing techniques. Despite eDNA analysis is a cost-effective non-invasive method for monitoring biodiversity, there's still a need to investigate factors affecting final outputs. We explored protocols for each step of the eDNA analysis, and mock communities were created to delve deeper into the variables influencing results. 13 DNA fish extracts were used to compose non-normalized (MC1) and normalized (MC2) mock communities. To investigate the mitochondrial DNA (mtDNA) proportion in fish extracts, MC1 and MC2 were created with pre-PCR extracts (before the 1st step PCR of the library preparation) and amplified extracts (post-PCR) by using tele02 primers targeting the 12S ribosomal RNA (rRNA) gene. The DNA library was prepared with a two-step approach, samples were pooled after indexing and sequenced on MiSeq Illumina (2X250PE). Demultiplexed sequences were processed with the ObiTools pipeline launched in Galaxy.eu. The parameters were set based on known species composition. We reported a significant and positive correlation (Pearson=0.9, p 0.05) between the number of reads and concentration only when the target gene is available in a high number of copies (MC1 post-PCR). Additionally, as expected, the Relative Read Abundance (RRA) pointed out an even proportion among species in MC2 (E=0.91) and MC1 (E=0.90) post-PCR, in contrast with a lack of evenness in MC2 (E=0.68) and MC1 (E=0.49) pre-PCR. We also highlighted that primer efficiency may vary among fish extracts and depending on starting concentrations, as no reads were assigned to Mullus barbatus in MC1 pre-PCR regardless of the starting concentration (13 ng/μl). Conversely, M. barbatus showed 10% RRA in MC1 post-PCR (averaged RRA/species was 7.86%). These results suggest that in real communities some species are less likely to be detected due to mtDNA starting concentration and primer efficiency.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
