Could Bento Lab be used to identify bats from their droppings using PCR? Some thoughts…

Could Bento Lab be used to identify bats from their droppings using DNA?

We don’t know of any researchers who are using Bento Lab to identify bats at the moment (if you are, or if you’d really like to do this, please let us know!).

But I do love the idea of Bento Lab being used by ecologists and bat conservationists to help survey and protect bats using DNA from their droppings!

So here are a few thoughts on:

• Why PCR-based bat identification is important
• How researchers are doing this work now
• How it might be done with Bento Lab
• And some links to recent research in the field

Why is bat identification important?

• Bats comprise ~20% of mammal species, with ~1,400 species currently recognised, and they are vital components of many ecosystems.
• An estimated 15% of bats (~200 species) are threatened with extinction due to human activity, and many species are very data deficient in terms of their population estimates and biology. 
• Bat populations in North America have also been severely impacted by the rise of White Nose Disease caused by the fungus Pseudogymnoascus destructans.
• Bats and their roosts are also legally protected in many countries, such as in most countries in Europe and the United Kingdom, wherever they may roost. In North America bats have protection in their natural habitats.
• You can read more about why bats are important at Batlife Europe, Bat Conservation International, and other bat conservation charities worldwide. 

The current state of research

There has been a lot of research into identification of bats over the past 10+ years. This means that there are some great articles to check out for potential protocols and primers. The studies below particularly caught my eye.

Hamilton et al. (2015) produced a PCR assay that could identify 15 species of British and Irish bats when paired with capillary electrophoresis. The assay uses a primer mix of 20 primers: five fluorescently tagged for detection by capillary electrophoresis, and 15 species-specific primers. Together these amplify one or more species-specific amplicons and a control amplicon for each sample, and the size of these amplicons are unique to each species. A really nice approach, but it is unfortunately not suitable for use with Bento Lab since the amplicons are very similar in size and wouldn’t visualise well enough on a small gel electrophoresis unit.

Walker et al. (2016) described a protocol for DNA barcoding of bats from their fecal pellets. This method is compatible with conventional PCR and Sanger sequencing, and also when paired with metabarcoding using Illumina MiSeq next generation sequencing. This protocol would be worth exploring for use with Bento Lab to produce DNA barcodes from single droppings via Sanger sequencing, and for use with Oxford Nanopore MinION barcoding for high-throughput metabarcoding.

The above protocol was revisited three years later by Walker et al. (2019) to test the limits of the method. They found that it worked well with faeces of up to two years in cool and dry environments, but DNA degraded by 12 months in extremely high humidity conditions. They also reported that this assay is now used by governmental agencies, research groups, and non-governmental organisations worldwide to identify bat species from guano!

Harrington et al. (2019) describe a qPCR protocol that could be used to identify British and Irish bats from their pellets. This uses qPCR and species-specific primers to amplify a very short (~80 bp) region of DNA from target species. The very short amplicons make it much easier to amplify bat DNA from the pellets, because shorter fragments would be less degraded than longer fragments. Very elegant, high throughput, and possibly much lower cost than Illumina metabarcoding, but this approach is unfortunately probably only suitable for qPCR devices.

Guan et al. (2020) reviewed the current literature, and described a new workflow and primer set from guano-derived DNA that can be used to identify nearly all US and Canadian bats to the species level, although some species remain difficult. The authors suggested that use of multiple protocols would have a better chance of detecting difficult species and could be easily done in parallel with little additional cost via metabarcoding.

A possible Bento Lab workflow for bat identification?

DNA barcoding from pellets via PCR and Sanger Sequencing

DNA barcoding from single pellets should be the easiest and achievable method to use outside of academic or industrial research. It would require:

• A DNA extraction method capable of extracting DNA from droppings. Walker et al. (2016) and Guan et al. (2020) used Qiagen’s DNeasy Blood and Tissue Kit, with the Animal Tissue Spin-Column protocol and a 12 hr tissue lysis. Other kits in the literature include the Zymo Research Genomic DNA^TM Tissue MicroPrep, QIAamp DNA Stool Mini Kit, PowerSoil DNA Isolation Kits. So probably any standard tissue or stool kit would be worth testing. Sourcing 100% or 97% ethanol to add to the kits will probably be required.
• Standard PCR equipment and reagents (a Bento Lab, PCR reagents, pipettes, plastics consumables)
• Bat barcoding primers from Walker et al. (2016).
• Access to a Sanger sequencing account.
• Basic knowledge and skills to curate and analyse sequences and to match them to a reference sequence database.

Costs per sample (for the UK) could be in the range of £2 per extraction and PCR, and £5-£10 per sequence (depending on whether they are single direction or a consensus sequence of forward and reverse sequences). This could offer a considerable saving compared to commercial services (which seem to range between £35 and £55 per sample in the UK), but it does exclude operator time and initial investment of equipment, learning of the workflow, and unexpected failures. Whether this is economically viable or not compared to commercial services will depend on economies of scale and operator expertise, and whether volunteers would be interested in doing this on a voluntary unpaid basis.

It might also be important to consider whether results provided by an individual would be sufficient for the purpose of the identification. For example, in some scenarios a result provided by an accredited commercial service for an ecological survey for building development planning purposes may be considered more reliable (or indeed the only valid option) compared to an individual’s or ecologist’s in-house sequencing results.

Nevertheless, a workflow along these lines could be really useful as part of a package of PCR and DNA barcoding tools for biodiversity research, where initial setup and training costs (including time) are spread across a range of different applications or interests, including bat DNA barcoding from pellets. For example, it could be ideal for pan-species DNA barcoding purposes, and especially during remote fieldwork.

We know from other research areas (especially fungal barcoding) that amateur experts are capable of learning and applying these workflows to an extremely high standard, so it would certainly be possible or similar work to be done on bat DNA barcoding from pellets, given access to the technology and enough time and training to learn.

This pellet-to-PCR-to-Sanger sequence workflow could also be useful in other forms of biodiversity research, such as in student or undergraduate and postgraduate research projects, or amateur bat surveillance, or public engagement. And its portability (except for Sanger sequencing, which would have to be done via a commercial or institutional service) could make it suitable for remote fieldwork or low resource contexts.

DNA metabarcoding from pellets

DNA metabarcoding from bat pellets is a much more specialised workflow and more suited to academic researchers at the moment, but it is conceivable (likely?) that advances in technology (hardware/software) and accessibility to that technology will improve a lot over the next ten years.

Researchers are already doing DNA metabarcoding from bat pellets with Illumina MiSeq sequencing for identification and diet analysis purposes , but other applications of metabarcoding in health/food safety/biodiversity studies are being done in a very portable and low cost way with Oxford Nanopore MinION sequencing, and there is no reason why the metabarcoding workflows couldn’t be easily adapted to work with MinION.

As has been seen with other studies, a Bento Lab-MinION barcoding and metabarcoding approach could be very useful in a range of applications, such as remote fieldwork, use in low resource research settings, or in time-critical research. This could allow a quick turnaround of results (same day or next day), at lower costs.

The current state of bat metabarcoding from pellets appears to be a robust and reliable process but one suitable primarily for academics/researchers/industry professionals.

But when/if technology and accessibility improves in the near future, it could have a lot of promise to become an exciting avenue of research by a wider community of practitioners, from professional ecologists to student projects, amateur research, or even secondary school education!

Do you have any thoughts on this idea, or would you be interested in trying out DNA barcoding of bats with Bento Lab? If you do, or if you have any comments, please get in touch!