Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints.

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

The environment and species affect gut bacteria composition in laboratory co-cultured Anopheles gambiae and Aedes albopictus mosquitoes.

The midgut microbiota of disease vectors plays a critical role in the successful transmission of human pathogens. The environment influences the microbiota composition; however, the relative mosquito-species contribution has not been rigorously disentangled from the environmental contribution to the microbiota structure. Also, the extent to which the microbiota of the adult sugar food source and larval water can predict that of the adult midgut and vice versa is not fully understood. To address these relationships, larvae and adults of Anopheles gambiae and Aedes albopictus were either reared separately or in a co-rearing system, whereby aquatic and adult stages of both species shared the larval water and sugar food source, respectively. Despite being reared under identical conditions, clear intra- and interspecies differences in midgut microbiota-composition were observed across seven cohorts, collected at different time points over a period of eight months. Fitting a linear model separately for each OTU in the mosquito midgut showed that two OTUs significantly differed between the midguts of the two mosquito species. We also show an effect for the sugar food source and larval water on the adult midgut microbiota. Our findings suggest that the mosquito midgut microbiota is highly dynamic and controlled by multiple factors.

Evaluation of the benefit of emergency vaccination in a foot-and-mouth disease free country with low livestock density.

Foot-and-mouth disease (FMD) is highly contagious and one of the most economically devastating diseases of cloven-hoofed animals. Scientific-based preparedness about how to best control the disease in a previously FMD-free country is therefore essential for veterinary services. The present study used a spatial, stochastic epidemic simulation model to compare the effectiveness of emergency vaccination with conventional (non-vaccination) control measures in Switzerland, a low-livestock density country. Model results revealed that emergency vaccination with a radius of 3 km or 10 km around infected premises (IP) did not significantly reduce either the cumulative herd incidence or epidemic duration if started in a small epidemic situation where the number of IPs is still low. However, in a situation where the epidemic has become extensive, both the cumulative herd incidence and epidemic duration are reduced significantly if vaccination were implemented with a radius of 10 km around IPs. The effect of different levels of conventional strategy measures was also explored for the non-vaccination strategy. It was found that a lower compliance level of farmers for movement restrictions and delayed culling of IPs significantly increased both the cumulative IP incidence and epidemic duration. Contingency management should therefore focus mainly on improving conventional strategies, by increasing disease awareness and communication with stakeholders and preparedness of culling teams in countries with a livestock structure similar to Switzerland; however, emergency vaccination should be considered if there are reasons to believe that the epidemic may become extensive, such as when disease detection has been delayed and many IPs are discovered at the beginning of the epidemic.

Re-establishment of local populations of vectors of Chagas disease after insecticide spraying.

1. Prevention of Chagas disease is mainly dependent on control of the insect vectors that transmit infection. Unfortunately, this control is not wholly successful and the vectors have been resurgent in some areas. Where re-infestation has occurred, it is important to understand the dynamics of the process. We investigated how a metapopulation framework can elucidate key aspects of re-infestation and thereby contribute to more efficient disease control.2.Triatoma infestans, the main vector of Chagas disease, re-infested sites in three villages in north-west Argentina after community-wide insecticide spraying in October 1992. Ten surveys were carried out at 6-monthly intervals from November 1994 to May 1999.3. Comparisons were made of different methods of estimating the sources of dispersal and the number of sites in which bug infestations became established.4. The results indicated that (i) the number of dispersing Triatoma infestans from a given site was proportional to the number of bugs found at the site; (ii) there was a 6-month time lag between detection of a new infestation and dispersal events; (iii) the relationship between infestations and new establishments varied by season.5. Three of 156 sites at which bugs were found were estimated to be the source of more than 50% of establishment events. These three sites were the only ones with large, persistent bug populations.6.Synthesis and applications. To reduce the risk of human Chagas disease, identifying those few sites infested with large, persistent bug populations and targeting control measures at those sites should greatly improve the efficiency of vector control. The appropriate seasonal timing of vector control could also greatly increase its efficiency. Specific recommendations for the timing of insecticide spraying require further research to establish how the observed temporal pattern of bug establishment is associated with the seasonality of bug dispersal.