C. Ryan Campbell

The Characterization of Twenty Sequenced Human Genomes

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

Exome Sequencing Followed by Large-Scale Genotyping Suggests a Limited Role for Moderately Rare Risk Factors of Strong Effect in Schizophrenia

Schizophrenia is a severe psychiatric disorder with strong heritability and marked heterogeneity in symptoms, course, and treatment response. There is strong interest in identifying genetic risk factors that can help to elucidate the pathophysiology and that might result in the development of improved treatments. Linkage and genome-wide association studies (GWASs) suggest that the genetic basis of schizophrenia is heterogeneous. However, it remains unclear whether the underlying genetic variants are mostly moderately rare and can be identified by the genotyping of variants observed in sequenced cases in large follow-up cohorts or whether they will typically be much rarer and therefore more effectively identified by gene-based methods that seek to combine candidate variants. Here, we consider 166 persons who have schizophrenia or schizoaffective disorder and who have had either their genomes or their exomes sequenced to high coverage. From these data, we selected 5,155 variants that were further evaluated in an independent cohort of 2,617 cases and 1,800 controls. No single variant showed a study-wide significant association in the initial or follow-up cohorts. However, we identified a number of case-specific variants, some of which might be real risk factors for schizophrenia, and these can be readily interrogated in other data sets. Our results indicate that schizophrenia risk is unlikely to be predominantly influenced by variants just outside the range detectable by GWASs. Rather, multiple rarer genetic variants must contribute substantially to the predisposition to schizophrenia, suggesting that both very large sample sizes and gene-based association tests will be required for securely identifying genetic risk factors.

Geogenetic patterns in mouse lemurs (genus Microcebus) reveal the ghosts of Madagascar's forests past

Phylogeographic analysis can be described as the study of the geological and climatological processes that have produced contemporary geographic distributions of populations and species. Here, we attempt to understand how the dynamic process of landscape change on Madagascar has shaped the distribution of a targeted clade of mouse lemurs (genus Microcebus) and, conversely, how phylogenetic and population genetic patterns in these small primates can reciprocally advance our understanding of Madagascars prehuman environment. The degree to which human activity has impacted the natural plant communities of Madagascar is of critical and enduring interest. Today, the eastern rainforests are separated from the dry deciduous forests of the west by a large expanse of presumed anthropogenic grassland savanna, dominated by the Family Poaceae, that blankets most of the Central Highlands. Although there is firm consensus that anthropogenic activities have transformed the original vegetation through agricultural and pastoral practices, the degree to which closed-canopy forest extended from the east to the west remains debated. Phylogenetic and population genetic patterns in a five-species clade of mouse lemurs suggest that longitudinal dispersal across the island was readily achieved throughout the Pleistocene, apparently ending at ∼55 ka. By examining patterns of both inter- and intraspecific genetic diversity in mouse lemur species found in the eastern, western, and Central Highland zones, we conclude that the natural environment of the Central Highlands would have been mosaic, consisting of a matrix of wooded savanna that formed a transitional zone between the extremes of humid eastern and dry western forest types.

Molecular evolutionary characterization of a V1R subfamily unique to strepsirrhine primates.

Vomeronasal receptor genes have frequently been invoked as integral to the establishment and maintenance of species boundaries among mammals due to the elaborate one-to-one correspondence between semiochemical signals and neuronal sensory inputs. Here, we report the most extensive sample of vomeronasal receptor class 1 (V1R) sequences ever generated for a diverse yet phylogenetically coherent group of mammals, the tooth-combed primates (suborder Strepsirrhini). Phylogenetic analysis confirms our intensive sampling from a single V1R subfamily, apparently unique to the strepsirrhine primates. We designate this subfamily as V1Rstrep. The subfamily retains extensive repertoires of gene copies that descend from an ancestral gene duplication that appears to have occurred prior to the diversification of all lemuriform primates excluding the basal genus Daubentonia (the aye-aye). We refer to the descendent clades as V1Rstrep-α and V1Rstrep-β. Comparison of the two clades reveals different amino acid compositions corresponding to the predicted ligand-binding site and thus potentially to altered functional profiles between the two. In agreement with previous studies of the mouse lemur (genus, Microcebus), the majority of V1Rstrep gene copies appear to be intact and under strong positive selection, particularly within transmembrane regions. Finally, despite the surprisingly high number of gene copies identified in this study, it is nonetheless probable that V1R diversity remains underestimated in these nonmodel primates and that complete characterization will be limited until high-coverage assembled genomes are available.

Assessing the utility of whole genome amplified DNA for next-generation molecular ecology

DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low‐quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next‐generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double‐digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturers recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de‐novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de‐novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next‐generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq

BackgroundRNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.ResultsWe found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.ConclusionsTranscriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.

The effect of body mass and diet composition on torpor patterns in a Malagasy primate (Microcebus murinus)

One of the most obvious physiological changes accompanying seasonal heterothermy in mammals is a fattening stage preceding periods of resource scarcity. This phenomenon reflects the interplay of both diet and physiology. Though the accrual of fat stores is known to be essential for overwintering in some species, the influence of diet on the physiology of torpor is not fully understood. Results from captive studies in heterothermic rodents and marsupials have indicated that when autumn diets are enriched with polyunsaturated fatty acids (PUFAs), animals receiving these diets experience deeper and more frequent torpor bouts than their counterparts receiving a control diet. Our study investigates this potential effect of dietary composition in animals that use daily torpor rather than prolonged torpor (i.e., hibernation). In so doing, we investigate the degree to which dietary effects on torpor are restricted to cold-adapted rodents and marsupials, or are a more general feature of mammalian heterothermy. We examined the effects of a PUFA diet and a control diet on the thermoregulation of one of the few species of primates known to use daily torpor: the grey mouse lemur (Microcebus murinus). Though the results of this study are largely inconclusive regarding the impact of dietary manipulations on torpor frequency and duration, we nonetheless find that the propensity of animals to enter torpor is directly influenced by age and seasonal changes in body mass, and thus reflect important physiological aspects of flexible thermoregulatory responses.

Evaluating whole genome sequence data from the Genetic Absence Epilepsy Rat from Strasbourg and its related non-epileptic strain

Objective The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbreed Wistar rat strain widely used as a model of genetic generalised epilepsy with absence seizures. As in humans, the genetic architecture that results in genetic generalized epilepsy in GAERS is poorly understood. Here we present the strain-specific variants found among the epileptic GAERS and their related Non-Epileptic Control (NEC) strain. The GAERS and NEC represent a powerful opportunity to identify neurobiological factors that are associated with the genetic generalised epilepsy phenotype. Methods We performed whole genome sequencing on adult epileptic GAERS and adult NEC rats, a strain derived from the same original Wistar colony. We also generated whole genome sequencing on four double-crossed (GAERS with NEC) F2 selected for high-seizing (n = 2) and non-seizing (n = 2) phenotypes. Results Specific to the GAERS genome, we identified 1.12 million single nucleotide variants, 296.5K short insertion-deletions, and 354 putative copy number variants that result in complete or partial loss/duplication of 41 genes. Of the GAERS-specific variants that met high quality criteria, 25 are annotated as stop codon gain/loss, 56 as putative essential splice sites, and 56 indels are predicted to result in a frameshift. Subsequent screening against the two F2 progeny sequenced for having the highest and two F2 progeny for having the lowest seizure burden identified only the selected Cacna1h GAERS-private protein-coding variant as exclusively co-segregating with the two high-seizing F2 rats. Significance This study highlights an approach for using whole genome sequencing to narrow down to a manageable candidate list of genetic variants in a complex genetic epilepsy animal model, and suggests utility of this sequencing design to investigate other spontaneously occurring animal models of human disease.