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A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.

ABSTRACT: Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5'-CGC GGC GCT ACA TAT TAG AC-3', Reverse: 5'-GCA CTT AAC CGA CCG TCG AC-3'). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44-66% of cephalopod species, corresponding to about 310-460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268-271; Peters et al. 2015 Mar. Ecol. 36, 1428-1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.

SUBMITTER: de Jonge DSW

PROVIDER: S-EPMC8074623 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although complete mitochondrial genome sequences have been reported for more than 600 animals, the taxonomic sampling is highly biased toward vertebrates and arthropods, leaving much of the diversity yet uncharacterized.ResultsThe mitochondrial genome of the bellybutton nautilus, Nautilus macromphalus, a cephalopod mollusk, is 16,258 nts in length and 59.5% A+T, both values that are typical of animal mitochondrial genomes. It contains the 37 genes that are almost universally found in animal mtDNAs, with 15 on one DNA strand and 22 on the other. The arrangement of these genes can be derived from that of the distantly related Katharina tunicata (Mollusca: Polyplacophora) by a switch in position of two large blocks of genes and transpositions of four tRNA genes. There is strong skew in the distribution of nucleotides between the two strands, and analysis of this yields insight into modes of transcription and replication. There is an unusual number of non-coding regions and their function, if any, is not known; however, several of these demark abrupt shifts in nucleotide skew, and there are several identical sequence elements at these junctions, suggesting that they may play roles in transcription and/or replication. One of the non-coding regions contains multiple repeats of a tRNA-like sequence. Some of the tRNA genes appear to overlap on the same strand, but this could be resolved if the polycistron were cleaved at the beginning of the downstream gene, followed by polyadenylation of the product of the upstream gene to form a fully paired structure.ConclusionNautilus macromphalus mtDNA contains an expected gene content that has experienced few rearrangements since the evolutionary split between cephalopods and polyplacophorans. It contains an unusual number of non-coding regions, especially considering that these otherwise often are generated by the same processes that produce gene rearrangements. The skew in nucleotide composition between the two strands is strong and associated with the direction of transcription in various parts of the genomes, but a comparison with K. tunicata implies that mutational bias during replication also plays a role. This appears to be yet another case where polyadenylation of mitochondrial tRNAs restores what would otherwise be an incomplete structure.

Project description:The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer "Sauron-S878," specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3' region, up to three mismatches in remaining primer). When paired with the reverse primer "jgHCO2198" (fragment length ~313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single-tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re-amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single-tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated.

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A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.

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