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 chloroplast
- NSF-PAR ID:
- 10314003
- Publisher / Repository:
- Dryad
- Date Published:
- Edition / Version:
- 3
- Subject(s) / Keyword(s):
- ["FOS: Biological sciences"]
- Format(s):
- Medium: X Size: 124634 bytes
- Size(s):
- ["124634 bytes"]
- Sponsoring Org:
- National Science Foundation
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Abstract trn L‐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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Rationale Protein studies in archaeology and paleontology have been dominated by stable isotope studies to understand diet and trophic levels, but recent applications of proteomic techniques have resulted in a more complete understanding of protein diagenesis than stable isotopes alone. In stable isotope analyses, samples are retained or discarded based on their properties. Proteomics can directly determine what proteins are present within the sample and may be able to allow previously discarded samples to be analyzed.
Methods Protein samples that had been previously analyzed for stable isotopes, including those with marginal and poor sample quality, were characterized by liquid chromatography/mass spectrometry using an LTQ Orbitrap Velos mass spectrometer after separation on a Dionex Ultimate 3000 LC system. Data were analyzed using MetaMorpheus and custom R scripts.
Results We found a variety of proteins in addition to collagen, although collagen I was found in the majority of the samples (most samples >80%). We also found a positive correlation between total deamidation and wt% N, suggesting that deamidation may impact the overall nitrogen signal in bulk analyses. The amino acid profiles of samples, including those of marginal or poor stable isotope quality, reflect the expected collagen I percentages, allowing their use in single amino acid stable isotope analyses.
Conclusions All the samples regardless of quality were found to have high concentrations of collagen I, making interpretations of dietary routing based on collagen I reasonably valid. The amino acid profiles on the marginal and poor samples reflect an expected collagen I profile and allow these samples to be recovered for single amino acid analyses.
