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4 results for “primer efficiency”
Data from: A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding
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 3 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 is 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.
Data from: Scaling up DNA barcoding - primer sets for simple and cost efficient arthropod systematics by multiplex PCR and Illumina amplicon sequencing
1. The simplicity and cost efficiency of Illumina amplicon sequencing has greatly contributed to the advancement of DNA barcoding and metabarcoding applications. However, current amplicon sequencing based barcoding approaches are usually restricted to short, single-locus fragments, limiting their taxonomic and phylogenetic resolution. 2. Here, we establish a cost efficient and simple multiplex PCR protocol for arthropod systematics by Illumina amplicon sequencing. We introduce primer sets, including several new, generic primers, to reliably amplify nine loci across a wide range of arthropods. Using a diverse collection of arthropod species from 19 orders, we test loci for amplification efficiency and estimate the effect of cross-species amplification bias on taxon recovery from bulk community samples. We then explore the taxonomic and phylogenetic utility of the primer sets, focusing on a dataset of spiders that includes both deep and recent divergences. 3. The set of loci provides good phylogenetic support across a wide taxonomic spectrum, making it a useful addition to COI for resolving lineages within a comparative context. All loci recover sequences for the majority of arthropod taxa in separate PCRs. However, cross-species amplification bias in some primers prevents an exhaustive taxon recovery from bulk community samples. 4. Our protocol makes it possible to generate multilocus datasets for large numbers of arthropod taxa for a fraction of the price and workload of Sanger sequencing. This opens up the possibility for parallel phylogenetic and taxonomic analysis of large collections of arthropods, but also enables rapid exploratory analyses of target lineages. Primers for metabarcoding applications should be carefully evaluated for their performance in bulk community samples and chosen to minimize cross-species amplification bias.
Data from: A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding
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Data from: Scaling up DNA barcoding - primer sets for simple and cost efficient arthropod systematics by multiplex PCR and Illumina amplicon sequencing
Open the record for dataset details and reuse information.
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