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Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches.
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A CRISPR ‐based strategy for targeted sequencing in biodiversity science
Abstract Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages in some applications. We identified 54,837 CRISPR‐Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single‐ and mixed‐species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed‐species experiments, single‐species experiments yielded more on‐target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed‐species experiments yielded sufficient data to provide ≥48‐fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplasttrnL‐P6 marker. Prior work developed CRISPR‐based enrichment protocols for long‐read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short‐read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR‐based analyses of mixed‐species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.
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- Award ID(s):
- 2046797
- PAR ID:
- 10482796
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- ISSN:
- 1755-098X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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