Peters, L. | bioRxiv Science

bioRxiv2018

B cell defects observed in Nod2 knockout mice are a consequence of a Dock2 mutation frequently found in inbred strains

Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments, and contribute to challenges with reproducibility across institutions. Notably, C57BL/6NHsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of Dock2. Here, we demonstrate the presence of this Dock2 variant in the widely used Nod2-/- mice. NOD2 is a cytosolic innate immune receptor that has been the subject of intense investigation due to its association with inflammatory bowel disease (IBD) susceptibility. Consistent with a role of NOD2 in an immunological disorder, Nod2-/- mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells and B1a cells. However, we found that these effects are due to the Dock2 variant and are independent of Nod2 deletion. Despite originating from the same gene-targeted founder mice, Nod2-/- mice from another source did not harbor the Dock2 variant or B cell defects. Finally, we show that Dock2-/- mice display the same B cell defects as mice harboring the Dock2 variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in Nod2-/- mice. Our findings highlight the effects of confounding mutations from widely-used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

immunology↗

bioRxiv2017

Environmental DNA: a new low-cost monitoring tool for pathogens in salmonid aquaculture.

Sequencing of environmental DNA (eDNA-seq) is an emergent new monitoring tool that promises to facilitate the accurate and cost effective detection of species in environmental samples. eDNA monitoring is likely to have a major impact on the ability of salmonid aquaculture industry producers and their regulators to detect the presence and abundance of pathogens and other biological threats in the surrounding environment. However, for eDNA-seq to develop into a useful bio-monitoring tool it is necessary to (a) validate that sequence datasets derived from amplification of meta-barcoding markers reflect the true species identity and abundances in biological samples, and (b) establish a low-cost sequencing method to enable the bulk processing of environmental samples. In this study, we employed an elaborate experimental design whereby different combinations of five biological agents were crossed at three abundance levels and exposed to pre-filtered and normal seawater, prior to coarse filtering and then eDNA ultrafiltration of the resultant material. We then benchmarked the low-cost, scalable, Ion Torrent sequencing method against the current gold-standard Illumina platform for eDNAseq detection in aquaculture. Based on amplicon-seq of the 18S SSU rDNA v9 region, we found that Illumina and Ion Torrent were equally good in identifying the two parasite species (Lepeophtheirus salmonis and Paramoeba perurans), whereas the microalgae species Prymnesium parvum, Pseudo-nitzschia seriata and P. delicatissima could be assigned correctly only to the genus level. Illumina and Ion Torrent were also equally able to reflect community composition in our samples, whereas Ion Torrent was more sensitive in detecting species richness when the medium was unfiltered seawater. Both methods were able to reflect the correct abundances of 4 out of 5 species in samples from unfiltered seawater, despite the significant amount of background noise from both bacteria and eukaryotes. Our findings indicate that eDNA-seq offers significant potential in the monitoring of species harmful to aquaculture and for this purpose, the low-cost Ion Torrent sequencing is equally as accurate as Illumina.

genetics↗