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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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On the causes, consequences, and avoidance of PCR duplicates: Towards a theory of library complexity
Abstract Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD‐seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification‐related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.
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- Award ID(s):
- 1645087
- PAR ID:
- 10473795
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 23
- Issue:
- 6
- ISSN:
- 1755-098X
- Page Range / eLocation ID:
- 1299 to 1318
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1111/1755-0998.13800
