Isolation and characterization of 21 polymorphic microsatellite loci in the iconic Australian lungfish, Neoceratodus forsteri, using the Ion Torrent next-generation sequencing platform

We isolated and characterized 21 microsatellite loci in the vulnerable and iconic Australian lungfish, Neoceratodus forsteri. Loci were screened across eight individuals from the Burnett River and 40 individuals from the Pine River. Genetic diversity was low with between one and six alleles per locus within populations and a maximum expected heterozygosity of 0.774. These loci will now be available to assess effective population sizes and genetic structure in N. forsteri across its natural range in South East Queensland, Australia.

Assembly and annotation of a non-model gastropod (Nerita melanotragus) transcriptome: a comparison of De novo assemblers

Background The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Results Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. Conclusions We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms. Keywords: Nerita melanotragus; De novo assembly; Transcriptome; Heat shock protein; Ion torrent

Molecular genetic investigation of mitochondrial dysfunction in relation to migraine susceptibility

The aim of this research was to assess the role of genetic variation in mitochondrial function and how this relates to migraine pathophysiology. Using our unique Norfolk Island population, a custom in-house next generation sequencing methodology was developed. This data for the first time showed that there is a molecular genetic link between mitochondrial dysfunction and migraine susceptibility. This work has provided the foundation for further studies aimed at utilising the identified markers in improved migraine diagnostic and therapeutic strategies.

Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus)

Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478 bp and N50 length of 506 bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

A transcriptomic analysis of the kidney tissue of Tra catfish (pangasianodon hypophthalmus) reared in saline condition: De novo assembly, annotation, SNP discovery

Pangasianodon hypophthalmus is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The current study using Ion Torrent technology generated EST resources from the kidney for Tra catfish reared at a salinity level of 9 ppt. We obtained 2,623,929 reads after trimming and processing with an average length of 104 bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 29,940 contigs, and allowing identification of 5,710 putative genes when comppared with NCBI non-redundant database. A large number of single nucleotide polymorphisms (SNPs) were also detected. The sequence collection generated in our study represents the most comprehensive transcriptomic resource for P. hypophthalmus available to date.

Air ion concentrations in various urban outdoor environments

Atmospheric ions are produced by many natural and anthropogenic sources and their concentrations vary widely between different environments. There is very little information on their concentrations in different types of urban environments, how they compare across these environments and their dominant sources. In this study, we measured airborne concentrations of small ions, particles and net particle charge at 32 different outdoor sites in and around a major city in Australia and identified the main ion sources. Sites were classified into seven groups as follows: park, woodland, city centre, residential, freeway, power lines and power substation. Generally, parks were situated away from ion sources and represented the urban background value of about 270 ions cm-3. Median concentrations at all other groups were significantly higher than in the parks. We show that motor vehicles and power transmission systems are two major ion sources in urban areas. Power lines and substations constituted strong unipolar sources, while motor vehicle exhaust constituted strong bipolar sources. The small ion concentration in urban residential areas was about 960 cm-3. At sites where ion sources were co-located with particle sources, ion concentrations were inhibited due to the ion-particle attachment process. These results improved our understanding on air ion distribution and its interaction with particles in the urban outdoor environment.

The role of vegetation in enhancing radon concentration and ion production in the atmosphere

The role of ions in the production of atmospheric particles has gained wide interest due to their profound impact on climate. Away from anthropogenic sources, molecules are ionized by alpha radiation from radon exhaled from the ground and cosmic gamma radiation from space. These molecular ions quickly form into ‘cluster ions’, typically smaller than about 1.5 nm. Using our measurements and the published literature, we present evidence to show that cluster ion concentrations in forest areas are consistently higher than outside. Since alpha radiation cannot penetrate more than a few centimetres of soil, radon present deep in the ground cannot directly contribute to the measured cluster ion concentrations. We propose an additional mechanism whereby radon, which is water soluble, is brought up by trees and plants through the uptake of groundwater and released into the atmosphere by transpiration. We estimate that, in a forest comprising eucalyptus trees spaced 4m apart, approximately 28% of the radon in the air may be released by transpiration. Considering that 24% of the earth’s land area is still covered in forests; these findings have potentially important implications for atmospheric aerosol formation and climate.