Camila J. Mazzoni

MHC genotyping of non-model organisms using next-generation sequencing: a new methodology to deal with artefacts and allelic dropout.

BackgroundThe Major Histocompatibility Complex (MHC) is the most important genetic marker to study patterns of adaptive genetic variation determining pathogen resistance and associated life history decisions. It is used in many different research fields ranging from human medical, molecular evolutionary to functional biodiversity studies. Correct assessment of the individual allelic diversity pattern and the underlying structural sequence variation is the basic requirement to address the functional importance of MHC variability. Next-generation sequencing (NGS) technologies are likely to replace traditional genotyping methods to a great extent in the near future but first empirical studies strongly indicate the need for a rigorous quality control pipeline. Strict approaches for data validation and allele calling to distinguish true alleles from artefacts are required.ResultsWe developed the analytical methodology and validated a data processing procedure which can be applied to any organism. It allows the separation of true alleles from artefacts and the evaluation of genotyping reliability, which in addition to artefacts considers for the first time the possibility of allelic dropout due to unbalanced amplification efficiencies across alleles. Finally, we developed a method to assess the confidence level per genotype a-posteriori, which helps to decide which alleles and individuals should be included in any further downstream analyses. The latter method could also be used for optimizing experiment designs in the future.ConclusionsCombining our workflow with the study of amplification efficiency offers the chance for researchers to evaluate enormous amounts of NGS-generated data in great detail, improving confidence over the downstream analyses and subsequent applications.

Cyanobacterial diversity in the hot spring, pelagic and benthic habitats of a tropical soda lake

Hot springs and saline-alkaline lakes of East Africa are extreme habitats regarding temperature, or salinity and pH, respectively. This study examines whether divergent habitats of Lake Bogoria, Kenya, impacts cyanobacterial community structure. Samples from the hot springs, pelagic zone and sediment were analysed by light microscopy, multilocus 454-amplicons sequencing and metagenomics to compare the cyanobacterial diversity. Most of the phylogenetic lineages of Cyanobacteria occurred exclusively in the Bogoria hot springs suggesting a high degree of endemism. The prevalent phylotypes were mainly members of the Oscillatoriales (Leptolyngbya, Spirulina, Oscillatoria-like and Planktothricoides). The Chroococcales were represented by different clades of Synechococcus but not a single phylotype clustered with any of the lineages described earlier from different continents. In contrast, we found that the pelagic zone and the sediments were inhabited by only a few taxa, dominated by Arthrospira and Anabaenopsis. Arthrospira, the main food base of Lesser Flamingo, was detected in all three habitats by amplicons pyrosequencing, indicating its resilience and key role as a primary producer. Despite the close connection between the three habitats studied, the cyanobacterial communities in the hot springs and lake differed considerably, suggesting that they are unable to adapt to the extreme conditions of the neighbouring habitat.

A population genomic scan in Chorthippus grasshoppers unveils previously unknown phenotypic divergence.

Understanding the genetics of speciation and the processes that drive it is a central goal of evolutionary biology. Grasshoppers of the Chorthippus species group differ strongly in calling song (and corresponding female preferences) but are exceedingly similar in other characteristics such as morphology. Here, we performed a population genomic scan on three Chorthippus species (Chorthippus biguttulus, C. mollis and C. brunneus) to gain insight into the genes and processes involved in divergence and speciation in this group. Using an RNA‐seq approach, we examined functional variation between the species by calling SNPs for each of the three species pairs and using FST‐based approaches to identify outliers. We found approximately 1% of SNPs in each comparison to be outliers. Between 37% and 40% of these outliers were nonsynonymous SNPs (as opposed to a global level of 17%) indicating that we recovered loci under selection. Among the outliers were several genes that may be involved in song production and hearing as well as genes involved in other traits such as food preferences and metabolism. Differences in food preferences between species were confirmed with a behavioural experiment. This indicates that multiple phenotypic differences implicating multiple evolutionary processes (sexual selection and natural selection) are present between the species.

Molecular characterization of canine kobuvirus in wild carnivores and the domestic dog in Africa

Knowledge of Kobuvirus (Family Picornaviridae) infection in carnivores is limited and has not been described in domestic or wild carnivores in Africa. To fill this gap in knowledge we used RT-PCR to screen fresh feces from several African carnivores. We detected kobuvirus RNA in samples from domestic dog, golden jackal, side-striped jackal and spotted hyena. Using next generation sequencing we obtained one complete Kobuvirus genome sequence from each of these species. Our phylogenetic analyses revealed canine kobuvirus (CaKV) infection in all four species and placed CaKVs from Africa together and separately from CaKVs from elsewhere. Wild carnivore strains were more closely related to each other than to those from domestic dogs. We found that the secondary structure model of the IRES was similar to the Aichivirus-like IRES subclass and was conserved among African strains. We describe the first CaKVs from Africa and extend the known host range of CaKV.

A novel endogenous betaretrovirus in the common vampire bat (Desmodus rotundus) suggests multiple independent infection and cross-species transmission events

ABSTRACT The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation.

A novel endogenous betaretrovirus group characterized from polar bears (Ursus maritimus) and giant pandas (Ailuropoda melanoleuca).

Transcriptome analysis of polar bears (Ursus maritimus) yielded sequences with highest similarity to the human endogenous retrovirus group HERV-K(HML-2). Further analysis of the polar bear draft genome identified an endogenous betaretrovirus group comprising 26 proviral copies and 231 solo LTRs. Molecular dating indicates the group originated before the divergence of bears from a common ancestor but is not present in all carnivores. Closely related sequences were identified in the giant panda (Ailuropoda melanoleuca) and characterized from its genome. We have designated the polar bear and giant panda sequences U. maritimus endogenous retrovirus (UmaERV) and A. melanoleuca endogenous retrovirus (AmeERV), respectively. Phylogenetic analysis demonstrated that the bear virus group is nested within the HERV-K supergroup among bovine and bat endogenous retroviruses suggesting a complex evolutionary history within the HERV-K group. All individual remnants of proviral sequences contain numerous frameshifts and stop codons and thus, the virus is likely non-infectious.

Canine distemper virus in the Serengeti ecosystem: molecular adaptation to different carnivore species.

Was the 1993/1994 fatal canine distemper virus (CDV) epidemic in lions and spotted hyaenas in the Serengeti ecosystem caused by the recent spillover of a virulent domestic dog strain or one well adapted to these noncanids? We examine this question using sequence data from 13 ‘Serengeti’ strains including five complete genomes obtained between 1993 and 2011. Phylogenetic and haplotype network analyses reveal that strains from noncanids during the epidemic were more closely related to each other than to those from domestic or wild canids. All noncanid ‘Serengeti’ strains during the epidemic encoded: (1) one novel substitution G134S in the CDV‐V protein; and (2) the rare amino acid combination 519I/549H at two sites under positive selection in the region of the CDV‐H protein that binds to SLAM (CD 150) host cell receptors. Worldwide, only a few noncanid strains in the America II lineage encode CDV‐H 519I/549H. All canid ‘Serengeti’ strains during the epidemic coded CDV‐V 134G, and CDV‐H 519R/549Y, or 519R/549H. A functional assay of cell entry revealed the highest performance by CDV‐H proteins encoding 519I/549H in cells expressing lion SLAM receptors, and the highest performance by proteins encoding 519R/549Y, typical of dog strains worldwide, in cells expressing dog SLAM receptors. Our findings are consistent with an epidemic in lions and hyaenas caused by CDV variants better adapted to noncanids than canids, but not with the recent spillover of a dog strain. Our study reveals a greater complexity of CDV molecular epidemiology in multihost environments than previously thought.

High adaptive variability and virus-driven selection on major histocompatibility complex (MHC) genes in invasive wild rabbits in Australia

The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus cuniculus). During the first disease outbreaks, RHDV caused mortality rates of up to 97% and reduced Australian rabbit numbers to very low levels. However, recently increased genetic resistance to RHDV and strong population growth has been reported. Major histocompatibility complex (MHC) class I immune genes are important for immune responses against viruses, and a high MHC variability is thought to be crucial in adaptive processes under pathogen-driven selection. We asked whether strong population bottlenecks and presumed genetic drift would have led to low MHC variability in wild Australian rabbits, and if the retained MHC variability was enough to explain the increased resistance against RHD. Despite the past bottlenecks we found a relatively high number of MHC class I sequences distributed over 2–4 loci. We identified positive selection on putative antigen-binding sites of the MHC. We detected evidence for RHDV-driven selection as one MHC supertype was negatively associated with RHD survival, fitting expectations of frequency-dependent selection. Gene duplication and pathogen-driven selection are possible (and likely) mechanisms that maintained the adaptive potential of MHC genes in Australian rabbits. Our findings not only contribute to a better understanding of the evolution of invasive species, they are also important in the light of planned future rabbit biocontrol in Australia.

Whole genome sequencing and methylome analysis of the wild guinea pig

BackgroundDNA methylation is a heritable mechanism that acts in response to environmental changes, lifestyle and diseases by influencing gene expression in eukaryotes. Epigenetic studies of wild organisms are mandatory to understand their role in e.g. adaptational processes in the great variety of ecological niches. However, strategies to address those questions on a methylome scale are widely missing. In this study we present such a strategy and describe a whole genome sequence and methylome analysis of the wild guinea pig.ResultsWe generated a full Wild guinea pig (Cavia aperea) genome sequence with enhanced coverage of methylated regions, benefiting from the available sequence of the domesticated relative Cavia porcellus. This new genome sequence was then used as reference to map the sequence reads of bisulfite treated Wild guinea pig sequencing libraries to investigate DNA-methylation patterns at nucleotide-specific level, by using our here described method, named ‘DNA-enrichment-bisulfite-sequencing’ (MEBS). The results achieved using MEBS matched those of standard methods in other mammalian model species. The technique is cost efficient, and incorporates both methylation enrichment results and a nucleotide-specific resolution even without a whole genome sequence available. Thus MEBS can be easily applied to extend methylation enrichment studies to a nucleotide-specific level.ConclusionsThe approach is suited to study methylomes of not yet sequenced mammals at single nucleotide resolution. The strategy is transferable to other mammalian species by applying the nuclear genome sequence of a close relative. It is therefore of interest for studies on a variety of wild species trying to answer evolutionary, adaptational, ecological or medical questions by epigenetic mechanisms.

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei

Abstract Background For more than 25 years, the golden mussel, Limnoperna fortunei, has aggressively invaded South American freshwaters, having travelled more than 5000 km upstream across 5 countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it. Findings We assembled the 1.6-Gb genome into 20 548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60 717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina paired-end, mate-paired, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.