Discussion
Challenges associated with plastid contamination represent a major
concern in microbiome analyses (Beckers et al. 2016; Jackrelet al. 2017; Gaona et al. 2020). Our results indicate that
the use of DNA-PNA clamps significantly improves the microbiome
sequencing output of faecal samples obtained from species with a diet
harbouring a large amount of chloroplast and mitochondrial DNA. This
effect has also been shown by
Fitzpatricket al. (2018) in plant surface microbiomes; however, our study is
the first to test the usefulness of clamps in wildlife microbiome
studies relying on faecal pellets. Microbiome studies have recently been
growing at an accelerated pace. As we move away from model organisms,
the diets of the animals under study become more and more diverse, As a
rough estimate, 26% of bats and 33% of birds
(Ko et al.2014) follow a plant-based diet. Therefore, techniques that allow us to
bypass the remnant plant material in faecal samples are becoming more
and more important for microbiome studies.
One important factor to keep in mind when using PNA-DNA clamps is the
need to have some information about the diet of the study species.
PNA-DNA clamp specificity varies between groups. In our case, we had
previous knowledge that, in our study area, the diet of Tequila bats
consists of almost 100% columnar cacti, particularly from one species,
namely Carnigea gigantea (LV and MT, personal observation and
unpublished data). In the bat case, visual inspection of the faecal
pellets also revealed that a large percentage of the pellets was
undigested pollen grain clusters. This facilitated the development of
the cpPNA clamp thanks to the information available from other studies
(Fitzpatricket al. 2018).
Our technique allows the more cost-effective use of sequencing capacity.
By employing PNA-DNA clamps, we have been able to target the “true
microbiome” more directly and waste fewer reads related to by-products
from the diet of the animal. Having higher read numbers enables better
statistical power in the analysis and decreases data losses in the
subsequent steps in downstream processing. Even though the sequencing
price tag is becoming cheaper every day (NHGRI
2020), without the
PNA-DNA clamps, we would have had to double or triple or even increase
by ten-fold our sequencing depth to make the latter reasonable enough to
allow downstream analyses. The cost of the clamps varies between
providers but, in general, the use of the clamps will always be more
cost-effective than aiming at larger sequencing depth. With the
expansion of microbiome studies to non-model organisms, we believe that
additional tools like the one presented in this paper will streamline
the future advancement of the field.