CRISPR-Cas9-mediated host signal reduction for 18S metabarcoding of host-associated eukaryotes.

Metabarcoding-based methods for identification of host-associated eukaryotes have the potential to revolutionize parasitology and microbial ecology, yet significant technical challenges remain. In particular, highly abundant host reads can mask the presence of less-abundant target organisms, especially for sample types rich in host DNA (e.g., blood and tissues). Here, we present a new CRISPR-Cas9-mediated approach designed to reduce host signal by selective amplicon digestion, thus enriching clinical samples for eukaryotic endosymbiont sequences during metabarcoding. Our method achieves a nearly 76% increased efficiency in host signal reduction compared with no treatment and a nearly 60% increased efficiency in host signal reduction compared with the most commonly used published method. Furthermore, the application of our method to clinical samples allows for the detection of parasite infections that would otherwise have been missed.

VESPA: an optimized protocol for accurate metabarcoding-based characterization of vertebrate eukaryotic endosymbiont and parasite assemblages.

Protocols for characterizing taxonomic assemblages by deep sequencing of short DNA barcode regions (metabarcoding) have revolutionized our understanding of microbial communities and are standardized for bacteria, archaea, and fungi. Unfortunately, comparable methods for host-associated eukaryotes have lagged due to technical challenges. Despite 54 published studies, issues remain with primer complementarity, off-target amplification, and lack of external validation. Here, we present VESPA (Vertebrate Eukaryotic endoSymbiont and Parasite Analysis) primers and optimized metabarcoding protocol for host-associated eukaryotic community analysis. Using in silico prediction, panel PCR, engineered mock community standards, and clinical samples, we demonstrate VESPA to be more effective at resolving host-associated eukaryotic assemblages than previously published methods and to minimize off-target amplification. When applied to human and non-human primate samples, VESPA enables reconstruction of host-associated eukaryotic endosymbiont communities more accurately and at finer taxonomic resolution than microscopy. VESPA has the potential to advance basic and translational science on vertebrate eukaryotic endosymbiont communities, similar to achievements made for bacterial, archaeal, and fungal microbiomes.

Serum Virome of Southern Beaufort Sea polar bears (Ursus maritimus) during a period of rapid climate change.

Climate change affects the behavior, physiology and life history of many Arctic wildlife species. It can also influence the distribution and ecology of infectious agents. The southern Beaufort Sea (SB) subpopulation of polar bears (Ursus maritimus) has experienced dramatic behavioral changes due to retreating sea ice and other climate-related factors, but the effects of these changes on physiology and infection remain poorly understood. Using serum from polar bears sampled between 2004 and 2015 and metagenomic DNA sequencing, we identified 48 viruses, all of the family Anelloviridae. Anelloviruses are small, ubiquitous infectious agents with circular single-stranded DNA genomes that are not known to cause disease but, in humans, covary in diversity and load with immunological compromise. We therefore examined the usefulness of anelloviruses as biomarkers of polar bear physiological stress related to climate and habitat use. Polar bear anelloviruses sorted into two distinct clades on a phylogenetic tree, both of which also contained anelloviruses of giant pandas (Ailuropoda melanoleuca), another ursid. Neither anellovirus diversity nor load were associated with any demographic variables, behavioral factors or direct physiological measures. However, pairwise genetic distances between anelloviruses were positively correlated with pairwise differences in sampling date, suggesting that the polar bear "anellome" is evolving over time. These findings suggest that anelloviruses are not a sensitive indicator of polar physiological stress, but they do provide a baseline for evaluating future changes to polar bear viromes.