BackgroundOne of the concerns of assembling de novo transcriptomes is determining the amount of read sequences required to ensure a comprehensive coverage of genes expressed in a particular sample. In this report, we describe the use of Illumina paired-end RNA-Seq (PE RNA-Seq) reads from Hevea brasiliensis (rubber tree) bark to devise a transcript mapping approach for the estimation of the read amount needed for deep transcriptome coverage.FindingsWe 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.ConclusionsWe 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.
Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease of the tropics with high clinical mortality rates. To date, no vaccines are approved for melioidosis and current treatment relies on antibiotics. Conversely, common misdiagnosis and high pathogenicity of Bp hamper efforts to fight melioidosis. This bacterium can be isolated from a wide range of niches such as waterlogged fields, stagnant water bodies, salt water bodies and from human and animal clinical specimens. Although extensive studies have been undertaken to elucidate pathogenesis mechanisms of Bp, little is known about how a harmless soil bacterium adapts to different environmental conditions, in particular, the shift to a human host to become a highly virulent pathogen. The bacterium has a large genome encoding an armory of factors that assist the pathogen in surviving under stressful conditions and assuming its role as a deadly intracellular pathogen. This review presents an overview of what is currently known about how the pathogen adapts to different environments. With in-depth understanding of Bp adaptation and survival, more effective therapies for melioidosis can be developed by targeting related genes or proteins that play a major role in the bacteria's survival.
Burkholderia pseudomallei , a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei , but not Burkholderia thailandensis . Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.
Application of beneficial microbes offers an environmentally friendly alternative for mitigation of basal stem rot (BSR) disease in oil palm. However, the biocontrol mechanisms of Trichoderma against the pathogenic Ganoderma spp. which cause BSR are largely unknown at the molecular level. To identify the transcripts involved during induced systemic resistance (ISR), we analyzed the root transcriptomes of oil palm seedlings inoculated simultaneously with both G. boninense and T. harzianum, and un-inoculated oil palm seedlings, as well as those that were inoculated with either pathogenic or beneficial fungi. Our analyses revealed that the biocontrol mechanisms of T. harzianum against G. boninense involve modulation of genes related to biosynthesis of phytohormones (ethylene, MeJA and MeSA), antioxidant (lascorbate and myo-inositol) and unique secondary metabolites such as momilactone, cell wall metabolisms, and detoxification of phytotoxic compounds; in addition to its role as a biofertilizer which improves nutritional status of host plant. The outcomes of this study have fueled our understanding on the biocontrol mechanisms involving T. harizianum against G. boninense infection in oil palm roots.
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