Graeme Fox

Fungal succession in an in-vessel composting system characterized using 454 pyrosequencing

Fungi are known to have an important role in the composting process as degraders of recalcitrant materials such as cellulose and lignin. Previous attempts to study the diversity and succession of fungi in compost systems have relied on the use of culture-dependent analyses and low-resolution DNA-fingerprinting techniques, lacking the necessary depth to analyse such a rich ecosystem. In this study, 454 pyrosequencing was used to characterize the fungal community composition at the different stages of an in-vessel composting process. A complex succession of fungi was revealed, with 251 fungal OTUs identified throughout the monitoring period. The Ascomycota were the dominant phylum (82.5% of all sequences recovered), followed by the Basidiomycota (10.4%) and the subphylum Mucoromycotina (4.9%). In the starting materials and early stages of the process, yeast species from the Saccharomycetales were abundant, while in latter stages and in the high temperature regions of the pile, fungi from the orders Eurotiales, Sordariales, Mucorales, Agaricales and Microascales were the most prominent. This study provides an improved understanding of the fungal diversity occurring during the composting of municipal solid waste, and this knowledge can lead to the development of more efficient composting practices and a better evaluation of the end-product quality.

Absence of ancient DNA in sub-fossil insect inclusions preserved in 'Anthropocene' Colombian copal.

Insects preserved in copal, the sub-fossilized resin precursor of amber, have potential value in molecular ecological studies of recently-extinct species and of extant species that have never been collected as living specimens. The objective of the work reported in this paper was therefore to determine if ancient DNA is present in insects preserved in copal. We prepared DNA libraries from two stingless bees (Apidae: Meliponini: Trigonisca ameliae) preserved in ‘Anthropocene’ Colombian copal, dated to ‘post-Bomb’ and 10,612±62 cal yr BP, respectively, and obtained sequence reads using the GS Junior 454 System. Read numbers were low, but were significantly higher for DNA extracts prepared from crushed insects compared with extracts obtained by a non-destructive method. The younger specimen yielded sequence reads up to 535 nucleotides in length, but searches of these sequences against the nucleotide database revealed very few significant matches. None of these hits was to stingless bees though one read of 97 nucleotides aligned with two non-contiguous segments of the mitochondrial cytochrome oxidase subunit I gene of the East Asia bumblebee Bombus hypocrita. The most significant hit was for 452 nucleotides of a 470-nucleotide read that aligned with part of the genome of the root-nodulating bacterium Bradyrhizobium japonicum. The other significant hits were to proteobacteria and an actinomycete. Searches directed specifically at Apidae nucleotide sequences only gave short and insignificant alignments. All of the reads from the older specimen appeared to be artefacts. We were therefore unable to obtain any convincing evidence for the preservation of ancient DNA in either of the two copal inclusions that we studied, and conclude that DNA is not preserved in this type of material. Our results raise further doubts about claims of DNA extraction from fossil insects in amber, many millions of years older than copal.

Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform

BackgroundThe composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols..MethodsDNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME.ResultsThere was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms).ConclusionsThe results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.

Metabarcoding analysis of home composts reveals distinctive fungal communities with a high number of unassigned sequences

Home composting has been strongly advocated in the UK, Europe and North America to divert organic waste away from conventional waste processing. Despite this, little attention has been given to microbial communities and their diversity in these systems. In this study, we examined the diversity of fungal species in 10 different domestic composts by 454 tag-encoded pyrosequencing. We report the recovery of 478 different molecular operational taxonomic units (MOTUs) from the 10 composts with a mean of 176.7 ± 19.6 MOTUs per compost and a mean of 12.9 ± 3.8 unique MOTUs per sample. Microascales (17.21 %), Hypocreales (16.76 %), Sordariales (14.89 %), Eurotiales (11.25 %) and Mortierellales (7.38 %) were the dominant orders in the community, with Pseudallescheria (9.52 %), Penicillium (8.43 %), Mortierella (3.60 %) and Fusarium (3.31 %) being the most abundant genera. Fungal communities in home composts were substantially different to large-scale commercial composts, with thermophilic and thermotolerant fungi present in much lower numbers. Significantly, 46.2 % of all sequences were identified as uncultured fungi or could not be assigned above the family level, suggesting there are a high number of new genera and species in these environments still to be described.

A Galaxy-based bioinformatics pipeline for optimised, streamlined microsatellite development from Illumina next-generation sequencing data

Microsatellites are useful tools for ecologists and conservationist biologists, but are taxa-specific and traditionally expensive and time-consuming to develop. New methods using next-generation sequencing (NGS) have reduced these problems, but the plethora of software available for processing NGS data may cause confusion and difficulty for researchers new to the field of bioinformatics. We developed a bioinformatics pipeline for microsatellite development from Illumina paired-end sequences, which is packaged in the open-source bioinformatics tool Galaxy. This optimises and streamlines the design of a microsatellite panel and provides a user-friendly graphical user interface. The pipeline utilises existing programs along with our own novel program and wrappers to: quality-filter and trim reads (Trimmomatic); generate sequence quality reports (FastQC); identify potentially-amplifiable microsatellite loci (Pal_finder); design primers (Primer3); assemble pairs of reads to enhance marker amplification success rates (PANDAseq); and filter optimal loci (Pal_filter). The complete pipeline is freely available for use via a pre-configured Galaxy instance, accessible at https://palfinder.ls.manchester.ac.uk.

Genetic variability and ontogeny predict microbiome structure in a disease-challenged montane amphibian

Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among- population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases.

Rapid isolation and characterization of microsatellites in the critically endangered mountain bongo ( Tragelaphus eurycerus isaaci )

High-throughput sequencing tools promise to revolutionize many aspects of genetic research, e.g. by allowing the identification of functional adaptive genetic variation. However, the expense and expertise required to apply these tools to basic conservation questions is a challenge for applications outside academia, resulting in a so-called ‘conservation genomics gap’ (Shafer et al.2015). The conservation genetics paradigm is that, basic information about inbreeding and gene flow are often critical to inform conservation management of small populations (Ouborg et al.2010). This information is often needed quickly and ideally should be accessible to workers without special expertise in genomics (DeSalle and Amato 2004). While the inferential power of high-throughput sequencing to interrogate the genome is profound, the cost for population analysis is higher (though decreasing) than for traditional neutral markers. Thus, the use of neutral markers is still relevant in conservation applications. However, this assumes that neutral markers have been discovered and characterized for a given species of conservation concern, which is often untrue for nonmodel organisms. Here, we use a fast, cost-efficient, high-throughput sequencing method (Illumina MiSeq) to rapidly identify and characterize microsatellites in the mountain bongo (Tragelaphus eurycerus isaaci), which has a clear and timely conservation imperative but lacks any described neutral markers.