Multigenerational Tracking of Reef-Building Corals Using CRISPR-Cas9 induced Genetic Barcodes and eDNA Metabarcoding
https://www.biorxiv.org/content/10.1101/2025.09.11.675722v1
#CRISPR #DNAmetabarcoding #eDNA
Multigenerational Tracking of Reef-Building Corals Using CRISPR-Cas9 induced Genetic Barcodes and eDNA Metabarcoding
Widespread biodiversity loss driven by human activity has intensified global efforts to restore degraded ecosystems. Yet a key challenge remains: how to track restored individuals and their offspring over time and space to assess the true impact of restoration? This is especially pressing for coral reefs, which support a quarter of all marine species, are in severe decline globally, and are now the focus of growing restoration initiatives worldwide. Here, we demonstrate a novel approach that combines genome editing and environmental DNA (eDNA) monitoring to enable scalable, non-invasive tracking of individual corals and their offspring. Using CRISPR-Cas9, we introduced unique genetic barcodes into non-coding regions of the Acropora millepora genome. These barcodes - stable, heritable, and distributed across the genome - will allow for individual-level identification over multiple generations. We show that these barcodes can be reliably detected in surrounding seawater using eDNA metabarcoding, offering a powerful, non-destructive tool for tracking corals in situ. Together, this approach provides a proof-of-concept for precision monitoring of restoration outcomes, with broad applicability across species and ecosystems.
### Competing Interest Statement
The authors have declared no competing interest.
**OptimOTU: Taxonomically aware OTU clustering with optimized thresholds and a bioinformatics workflow for metabarcoding data**
https://arxiv.org/abs/2502.10350
#OTU #clustering #bioinformatics #DNAmetabarcoding
OptimOTU: Taxonomically aware OTU clustering with optimized thresholds and a bioinformatics workflow for metabarcoding data
To turn environmentally derived metabarcoding data into community matrices for ecological analysis, sequences must first be clustered into operational taxonomic units (OTUs). This task is particularly complex for data including large numbers of taxa with incomplete reference libraries. OptimOTU offers a taxonomically aware approach to OTU clustering. It uses a set of taxonomically identified reference sequences to choose optimal genetic distance thresholds for grouping each ancestor taxon into clusters which most closely match its descendant taxa. Then, query sequences are clustered according to preliminary taxonomic identifications and the optimized thresholds for their ancestor taxon. The process follows the taxonomic hierarchy, resulting in a full taxonomic classification of all the query sequences into named taxonomic groups as well as placeholder "pseudotaxa" which accommodate the sequences that could not be classified to a named taxon at the corresponding rank. The OptimOTU clustering algorithm is implemented as an R package, with computationally intensive steps implemented in C++ for speed, and incorporating open-source libraries for pairwise sequence alignment. Distances may also be calculated externally, and may be read from a UNIX pipe, allowing clustering of large datasets where the full distance matrix would be inconveniently large to store in memory. The OptimOTU bioinformatics pipeline includes a full workflow for paired-end Illumina sequencing data that incorporates quality filtering, denoising, artifact removal, taxonomic classification, and OTU clustering with OptimOTU. The OptimOTU pipeline is developed for use on high performance computing clusters, and scales to datasets with millions of reads per sample, and tens of thousands of samples.
**Desiccation as a suitable alternative to cold-storage of phyllosphere samples for DNA-based microbial community analyses**
https://www.nature.com/articles/s41598-024-82367-x
#eDNA #DNAmetabarcoding #phyllosphere #bacteria #fungi
Desiccation as a suitable alternative to cold-storage of phyllosphere samples for DNA-based microbial community analyses - Scientific Reports
The study of microbial communities of the plant phyllosphere in remote locations using DNA-based approaches is limited by the challenges associated with their preservation in the field and during transportation. Freezing is a common DNA preservation strategy, but it may be unsuitable for leaf samples, or inaccessible in some locations. Other methods such as desiccation, ethanol or commercial preservatives are potential alternative DNA preservation methods for ambient temperature storage. In this study, we assessed the efficacy of desiccation (with silica gel packs), and of three preservation solutions (95% ethanol, RNAlater, LifeGuard) for the preservation of epiphytic phyllosphere communities of Populus tremuloides and Picea glauca at ambient indoor temperature (21 Β°C) for up to three weeks. We assessed effects on DNA concentration and quality and used metabarcoding to detect changes in bacterial and fungal communities between treatments over time. A secondary study was conducted on leaves of Populus grandidentata to further test the ability of the desiccation treatment to resolve differences between sampling sites. Silica gel packs were identified as effective ambient temperature preservative of phyllosphere bacterial and fungal communities. There were some changes in the communities compared to immediate extraction due to this treatment, but these changes did not affect the ability to distinguish tree species and sampling locations. Overall, our study supports the use of silica gel pack short term preservation at ambient temperature for phyllosphere samples intended for DNA-based microbial community analyses.
**Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring**
https://mbmg.pensoft.net/article/133264
#DNAmetabarcoding #standardization #freshwater #biomonitoring
Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring
DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an βall for one but prove them allβ strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.
"ο»ΏProficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring"
https://mbmg.pensoft.net/article/133264
#DNAmetabarcoding #standardization #ProficiencyTesting #biomonitoring
Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoring
DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an βall for one but prove them allβ strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.
The MIEM guidelines: Minimum information for reporting of environmental metabarcoding data
https://mbmg.pensoft.net/article/128689/
#DNAmetabarcoding #eDNA #eRNA #FAIR #biodiversity
The MIEM guidelines: Minimum information for reporting of environmental metabarcoding data
Environmental DNA (eDNA) and RNA (eRNA) metabarcoding has become a popular tool for assessing biodiversity from environmental samples, but inconsistent documentation of methods, data and metadata makes results difficult to reproduce and synthesise. A working group of scientists have collaborated to produce a set of minimum reporting guidelines for the constituent steps of metabarcoding workflows, from the physical layout of laboratories through to data archiving. We emphasise how reporting the suite of data and metadata should adhere to findable, accessible, interoperable and reproducible (FAIR) data standards, thereby providing context for evaluating and understanding study results. An overview of the documentation considerations for each workflow step is presented and then summarised in a checklist that can accompany a published study or report. Ensuring workflows are transparent and documented is critical to reproducible research and should allow for more efficient uptake of metabarcoding data into management decision-making.
An overlooked source of false positives in eDNA-based biodiversity assessment and management
https://doi.org/10.1016/j.jenvman.2024.120949
"eDNA contamination in treated effluent was common across popular processes. Numerous commercial and ornamental fish were recovered from treated effluent. eDNA pollution can lead to false positives in biodiversity assessment."
#DNAmetabarcoding #eDNA #biodiversity #urban #aquatic #ecosystems
Teresita Porter ππ»ββοΈ