Evaluating single-cell cluster stability using the Jaccard similarity index.

MOTIVATION: One major goal of single-cell RNA sequencing (scRNAseq) experiments is to identify novel cell types. With increasingly large scRNAseq datasets, unsupervised clustering methods can now produce detailed catalogues of transcriptionally distinct groups of cells in a sample. However, the interpretation of these clusters is challenging for both technical and biological reasons. Popular clustering algorithms are sensitive to parameter choices, and can produce different clustering solutions with even small changes in the number of principal components used, the k nearest neighbor and the resolution parameters, among others. RESULTS: Here, we present a set of tools to evaluate cluster stability by subsampling, which can guide parameter choice and aid in biological interpretation. The R package scclusteval and the accompanying Snakemake workflow implement all steps of the pipeline: subsampling the cells, repeating the clustering with Seurat and estimation of cluster stability using the Jaccard similarity index and providing rich visualizations. AVAILABILITYAND IMPLEMENTATION: R package scclusteval: https://github.com/crazyhottommy/scclusteval Snakemake workflow: https://github.com/crazyhottommy/pyflow_seuratv3_parameter Tutorial: https://crazyhottommy.github.io/EvaluateSingleCellClustering/.

Trappc9 deficiency causes parent-of-origin dependent microcephaly and obesity.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.

Characterization of Genomic Variants Associated with Scout and Recruit Behavioral Castes in Honey Bees Using Whole-Genome Sequencing.

Among forager honey bees, scouts seek new resources and return to the colony, enlisting recruits to collect these resources. Differentially expressed genes between these behaviors and genetic variability in scouting phenotypes have been reported. Whole-genome sequencing of 44 Apis mellifera scouts and recruits was undertaken to detect variants and further understand the genetic architecture underlying the behavioral differences between scouts and recruits. The median coverage depth in recruits and scouts was 10.01 and 10.7 X, respectively. Representation of bacterial species among the unmapped reads reflected a more diverse microbiome in scouts than recruits. Overall, 1,412,705 polymorphic positions were analyzed for associations with scouting behavior, and 212 significant (p-value < 0.0001) associations with scouting corresponding to 137 positions were detected. Most frequent putative transcription factor binding sites proximal to significant variants included Broad-complex 4, Broad-complex 1, Hunchback, and CF2-II. Three variants associated with scouting were located within coding regions of ncRNAs including one codon change (LOC102653644) and 2 frameshift indels (LOC102654879 and LOC102655256). Significant variants were also identified on the 5'UTR of membrin, and 3'UTRs of laccase 2 and diacylglycerol kinase theta. The 60 significant variants located within introns corresponded to 39 genes and most of these positions were > 1000 bp apart from each other. A number of these variants were mapped to ncRNA LOC100578102, solute carrier family 12 member 6-like gene, and LOC100576965 (meprin and TRAF-C homology domain containing gene). Functional categories represented among the genes corresponding to significant variants included: neuronal function, exoskeleton, immune response, salivary gland development, and enzymatic food processing. These categories offer a glimpse into the molecular support to the behaviors of scouts and recruits. The level of association between genomic variants and scouting behavior observed in this study may be linked to the honey bee's genomic plasticity and fluidity of transition between castes.

Comparative brain transcriptomic analyses of scouting across distinct behavioural and ecological contexts in honeybees.

Individual differences in behaviour are often consistent across time and contexts, but it is not clear whether such consistency is reflected at the molecular level. We explored this issue by studying scouting in honeybees in two different behavioural and ecological contexts: finding new sources of floral food resources and finding a new nest site. Brain gene expression profiles in food-source and nest-site scouts showed a significant overlap, despite large expression differences associated with the two different contexts. Class prediction and 'leave-one-out' cross-validation analyses revealed that a bee's role as a scout in either context could be predicted with 92.5% success using 89 genes at minimum. We also found that genes related to four neurotransmitter systems were part of a shared brain molecular signature in both types of scouts, and the two types of scouts were more similar for genes related to glutamate and GABA than catecholamine or acetylcholine signalling. These results indicate that consistent behavioural tendencies across different ecological contexts involve a mixture of similarities and differences in brain gene expression.

The transcription factor ultraspiracle influences honey bee social behavior and behavior-related gene expression.

Behavior is among the most dynamic animal phenotypes, modulated by a variety of internal and external stimuli. Behavioral differences are associated with large-scale changes in gene expression, but little is known about how these changes are regulated. Here we show how a transcription factor (TF), ultraspiracle (usp; the insect homolog of the Retinoid X Receptor), working in complex transcriptional networks, can regulate behavioral plasticity and associated changes in gene expression. We first show that RNAi knockdown of USP in honey bee abdominal fat bodies delayed the transition from working in the hive (primarily "nursing" brood) to foraging outside. We then demonstrate through transcriptomics experiments that USP induced many maturation-related transcriptional changes in the fat bodies by mediating transcriptional responses to juvenile hormone. These maturation-related transcriptional responses to USP occurred without changes in USP's genomic binding sites, as revealed by ChIP-chip. Instead, behaviorally related gene expression is likely determined by combinatorial interactions between USP and other TFs whose cis-regulatory motifs were enriched at USP's binding sites. Many modules of JH- and maturation-related genes were co-regulated in both the fat body and brain, predicting that usp and cofactors influence shared transcriptional networks in both of these maturation-related tissues. Our findings demonstrate how "single gene effects" on behavioral plasticity can involve complex transcriptional networks, in both brain and peripheral tissues.

Molecular determinants of scouting behavior in honey bees.

Little is known about the molecular basis of differences in behavior among individuals. Here we report consistent novelty-seeking behavior, across different contexts, among honey bees in their tendency to scout for food sources and nest sites, and we reveal some of the molecular underpinnings of this behavior relative to foragers that do not scout. Food scouts showed extensive differences in brain gene expression relative to other foragers, including differences related to catecholamine, glutamate, and γ-aminobutyric acid signaling. Octopamine and glutamate treatments increased the likelihood of scouting, whereas dopamine antagonist treatment decreased it. These findings demonstrate intriguing similarities in human and insect novelty seeking and suggest that this trait, which presumably evolved independently in these two lineages, may be subserved by conserved molecular components.