Epigenetic ageing in a human context, has been used to better understand the relationship between age and factors such as lifestyle and genetics. In an ecological setting, it has been used to predict the age of individual animals for wildlife management. Despite the importance of epigenetic ageing in a range of research fields, the assays to measure epigenetic ageing are either expensive on a large scale or complex. In this study, we aimed to improve the efficiency and sequencing quality of an existing epigenetic ageing assay for the Australian Lungfish (Neoceratodus forsteri). We used an enzyme-based alternative to bisulfite conversion to reduce DNA fragmentation and evaluated its performance relative to bisulfite conversion. We found the sequencing quality to be 12% higher with the enzymatic alternative compared to bisulfite treatment (p-value < 0.01). This new enzymatic based approach, although currently double the cost of bisulfite treatment can increases the throughput and sequencing quality. We envisage this assay setup being adopted increasingly as the scope and scale of epigenetic ageing research continues to grow.
Aging , Epigenesis, Genetic , Sulfites , Animals , Aging/genetics , Sulfites/chemistry , Fishes/genetics , Sequence Analysis, DNA/methods , DNA Methylation , DNA Fragmentation
Next generation sequencing and bioinformatic approaches are increasingly used to quantify microorganisms within populations by analysis of 'meta-barcode' data. This approach relies on comparison of amplicon sequences of 'barcode' regions from a population with public-domain databases of reference sequences. However, for many organisms relevant 'barcode' regions may not have been identified and large databases of reference sequences may not be available. A workflow and software pipeline, 'MetaGaAP,' was developed to identify and quantify genotypes through four steps: shotgun sequencing and identification of polymorphisms in a metapopulation to identify custom 'barcode' regions of less than 30 polymorphisms within the span of a single 'read', amplification and sequencing of the 'barcode', generation of a custom database of polymorphisms, and quantitation of the relative abundance of genotypes. The pipeline and workflow were validated in a 'wild type' Alphabaculovirus isolate, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV-AC53) and a tissue-culture derived strain (HaSNPV-AC53-T2). The approach was validated by comparison of polymorphisms in amplicons and shotgun data, and by comparison of predicted dominant and co-dominant genotypes with Sanger sequences. The computational power required to generate and search the database effectively limits the number of polymorphisms that can be included in a barcode to 30 or less. The approach can be used in quantitative analysis of the ecology and pathology of non-model organisms.
Complete genome sequences of two Australian isolates of H. armigera single nucleopolyhedrovirus (HaSNPV) and nine strains isolated by plaque selection in tissue culture identified multiple polymorphisms in tissue culture-derived strains compared to the consensus sequence of the parent isolate. Nine open reading frames (ORFs) in all tissue culture-derived strains contained changes in nucleotide sequences that resulted in changes in predicted amino acid sequence compared to the parent isolate. Of these, changes in predicted amino acid sequence of six ORFs were identical in all nine derived strains. Comparison of sequences and maximum likelihood estimation (MLE) of specific ORFs and whole genome sequences were used to compare the isolates and derived strains to published sequence data from other HaSNPV isolates. The Australian isolates and derived strains had greater sequence similarity to New World SNPV isolates from H. zea than to Old World isolates from H. armigera, but with characteristics associated with both. Three distinct geographic clusters within HaSNPV genome sequences were identified: Australia/Americas, Europe/Africa/India, and China. Comparison of sequences and fragmentation of ORFs suggest that geographic movement and passage in vitro result in distinct patterns of baculovirus strain selection and evolution.
Genetic Variation , Lepidoptera/virology , Nucleopolyhedroviruses/genetics , Nucleopolyhedroviruses/isolation & purification , Animals , Australia , Cluster Analysis , Mutation, Missense , Nucleopolyhedroviruses/classification , Open Reading Frames , Sequence Analysis, DNA , Sequence Homology , Viral Plaque Assay , Virus Cultivation
Granuloviruses are widespread pathogens of Plutella xylostella L. (diamondback moth) and potential biopesticides for control of this global insect pest. We report the complete genomes of four Plutella xylostella granulovirus isolates from China, Malaysia, and Taiwan exhibiting pairs of noncoding, homologous repeat regions with significant sequence variation but equivalent length.
Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from Helicoverpa armigera SNPV-AC53 (KJ909666).
We report here the genome sequences of two alphabaculoviruses of Helicoverpa spp. from Australia: AC53, used in the biopesticides ViVUS and ViVUS Max, and H25EA1, used in in vitro production studies.
