FINDINGS: We optimized the assembly of a Hevea bark transcriptome based on 16 Gb Illumina PE RNA-Seq reads using the Oases assembler across a range of k-mer sizes. We then assessed assembly quality based on transcript N50 length and transcript mapping statistics in relation to (a) known Hevea cDNAs with complete open reading frames, (b) a set of core eukaryotic genes and (c) Hevea genome scaffolds. This was followed by a systematic transcript mapping process where sub-assemblies from a series of incremental amounts of bark transcripts were aligned to transcripts from the entire bark transcriptome assembly. The exercise served to relate read amounts to the degree of transcript mapping level, the latter being an indicator of the coverage of gene transcripts expressed in the sample. As read amounts or datasize increased toward 16 Gb, the number of transcripts mapped to the entire bark assembly approached saturation. A colour matrix was subsequently generated to illustrate sequencing depth requirement in relation to the degree of coverage of total sample transcripts.
CONCLUSIONS: We devised a procedure, the "transcript mapping saturation test", to estimate the amount of RNA-Seq reads needed for deep coverage of transcriptomes. For Hevea de novo assembly, we propose generating between 5-8 Gb reads, whereby around 90% transcript coverage could be achieved with optimized k-mers and transcript N50 length. The principle behind this methodology may also be applied to other non-model plants, or with reads from other second generation sequencing platforms.
Methods: Combination of throat and nasal swab specimens was subjected to viral RNA extraction. For screening, the extracted RNA was subjected to real-time RT-PCR targeting upstream of E gene, open reading frame 1b and open reading frame 1a. For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase and nucleocapsid, followed by amplification of complete N gene region. Nucleotide sequencing of the first Malaysian case of MERS-CoV was performed following the confirmation with real-time RT-PCR detection.
Results: Initial analysis of partial RNA-dependent RNA polymerase and N gene revealed that the nucleotides had high similarity to Jeddah_1_2013 strain. Analysis of complete N gene region (1 242 nucleotides) from the case showed high similarity and yet distinct to the nucleotide sequences of camel-derived MERS-CoV.
Conclusions: From the finding, there are possibilities that the patient acquired the infection from zoonotic transmission from dromedary camels.
RESULTS: A molecular phylogenetic analysis of the mitochondrial ORF and putative control region concurs with a haploweb analysis of nuclear ITS2 sequences in delimiting three species among our dataset: species A and B are found in Madagascar whereas species C occurs in Okinawa, the Philippines and New Caledonia. Comparison of ITS1 sequences from these three species with data available online suggests that species C is also found on the Great Barrier Reef, in Malaysia, in the South China Sea and in Taiwan, and that a distinct species D occurs in the Red Sea. Shallow-water morphs of species A correspond to the morphological description of Stylophora madagascarensis, species B presents the morphology of Stylophora mordax, whereas species C comprises various morphotypes including Stylophora pistillata and Stylophora mordax.
CONCLUSIONS: Genetic analysis of the coral genus Stylophora reveals species boundaries that are not congruent with morphological traits. Of the four hypotheses that may explain such discrepancy (phenotypic plasticity, morphological stasis, morphological convergence, and interspecific hybridization), the first two appear likely to play a role but the fourth one is rejected since mitochondrial and nuclear markers yield congruent species delimitations. The position of the root in our molecular phylogenies suggests that the center of origin of Stylophora is located in the western Indian Ocean, which probably explains why this genus presents a higher biodiversity in the westernmost part of its area of distribution than in the "Coral Triangle".