Microsatellite loci and the complete mitochondrial DNA sequence characterized through next generation sequencing and de novo genome assembly for the critically endangered orange-bellied parrot, Neophema chrysogaster

A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.

Characterisation of the complete mitochondrial genome and 13 microsatellite loci through next-generation sequencing for the New Caledonian spider-ant Leptomyrmex pallens

The complete mitochondrial genome and a set of polymorphic microsatellite markers were identified by 454 pyrosequencing (1/16th of a plate) for the New Caledonian rainforest spider-ant Leptomyrmex pallens. De novo genome assembly recovered the entire mitochondrial genome with mean coverage of 8.9-fold (range 1-27). The mitogenome consists of 15,591 base pairs including 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The genome arrangement is typical of insect taxa and very similar to the only other published ant mitogenome from the Solenopsis genus, with the main differences consisting of translocations and inversions of tRNAs. A total of 13 polymorphic loci were also characterized using 41 individuals from a single population in the Aoupinié region, corresponding to workers from 21 nests and 16 foraging workers. We observed moderate genetic variation across most loci (mean number of alleles per locus = 4.50; mean expected heterozygosity = 0.53) with evidence of only two loci deviating significantly from Hardy-Weinberg equilibrium due to null alleles. Marker independence was confirmed with tests for linkage disequilibrium. Most loci cross amplified for three additional Leptomyrmex species. The annotation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the colony structure, population genetic patterns, and dispersal strategy of L. pallens in the context of rainforest fragmentation in New Caledonia. Furthermore, this paper confirms a recent line of evidence that comprehensive mitochondrial data can be obtained relatively easily from small next-generation sequencing analyses. Greater synthesis of next-generation sequencing data will play a significant role in expanding the taxonomic representation of mitochondrial genome sequences.

De novo assembly and characterization of the invasive Northern Pacific Seastar transcriptome

Invasive species are a major threat to global biodiversity but can also serve as valuable model systems to examine important evolutionary processes. While the ecological aspects of invasions have been well documented, the genetic basis of adaptive change during the invasion process has been hampered by a lack of genomic resources for the majority of invasive species. Here we report the first larval transcriptomic resource for the Northern Pacific Seastar, Asterias amurensis, an invasive marine predator in Australia. Approximately 117.5 million 100 base-pair (bp) paired-end reads were sequenced from a single RNA-Seq library from a pooled set of full-sibling A. amurensis bipinnaria larvae. We evaluated the efficacy of a pre-assembly error correction pipeline on subsequent de novo assembly. Error correction resulted in small but important improvements to the final assembly in terms of mapping statistics and core eukaryotic genes representation. The error-corrected de novo assembly resulted in 115,654 contigs after redundancy clustering. 41,667 assembled contigs were homologous to sequences from NCBI's non-redundant protein and UniProt databases. We assigned Gene Ontology, KEGG Orthology, Pfam protein domain terms and predicted protein-coding sequences to > 36,000 contigs. The final transcriptome dataset generated here provides functional information for 18,319 unique proteins, comprising at least 11,355 expressed genes. Furthermore, we identified 9,739 orthologs to P. miniata proteins, evaluated our annotation pipeline and generated a list of 150 candidate genes for responses to several environmental stressors that may be important for adaptation of A. amurensis in the invasive range. Our study has produced a large set of A. amurensis RNA contigs with functional annotations that can serve as a resource for future comparisons to other echinoderm transcriptomes and gene expression studies. Our data can be used to study the genetic basis of adaptive change and other important evolutionary processes during a successful invasion.

A parallel Euler approach for large-scale biological sequence assembly

Biological sequence assembly is an essential step for sequencing the genomes of organisms. Sequence assembly is very computing intensive especially for the large-scale sequence assembly. Parallel computing is an effective way to reduce the computing time and support the assembly for large amount of biological fragments. Euler sequence assembly algorithm is an innovative algorithm proposed recently. The advantage of this algorithm is that its computing complexity is polynomial and it provides a better solution to the notorious “repeat” problem. This paper introduces the parallelization of the Euler sequence assembly algorithm. All the Genome fragments generated by whole genome shotgun (WGS) will be assembled as a whole rather than dividing them into groups which may incurs errors due to the inaccurate group partition. The implemented system can be run on supercomputers, network of workstations or even network of PC computers. The experimental results have demonstrated the performance of our system.

A parallel DNA fragment assembly algorithm based on eulerian superpath approach

Fragments assembly is among the core problems in the research of Genome. Although many assembly tools based on the "overlap-layout-consensus" paradigm are widely used such as in the Human Genome Project currently, they still can not resolve the "repeats problem" in the DNA sequencing. For the purpose of resolving such problem, Pevzner et al. put forward a new Euler Superpath assembly algorithm. But it needs a big and complex de Bruijin graph which consumes large amounts of memories i.e. becomes the bottleneck of the performance. We present a parallel DNA fragment assembly algorithm based on the Eularian Superpath theory and solve the bottleneck in the current assembly program. The experimental results demonstrate that our approach has a good scalability, and can be used in DNA assembly of middle and large size of eukaryote genome.

Apicoplast-localized lysophosphatidic acid precursor assembly is required for bulk phospholipid synthesis in toxoplasma gondii and relies on an algal/plant-like glycerol 3-phosphate acyltransferase

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.