Transcriptomic complexity of the human malaria parasite Plasmodium falciparum revealed by long-read sequencing.

The Plasmodium falciparum human malaria parasite genome is incompletely annotated and does not accurately represent the transcriptomic diversity of this species. To address this need, we performed long-read transcriptomic sequencing. 5' capped mRNA was enriched from samples of total and nuclear-fractionated RNA from intra-erythrocytic stages and converted to cDNA library. The cDNA libraries were sequenced on PacBio and Nanopore long-read platforms. 12,495 novel isoforms were annotated from the data. Alternative 5' and 3' ends represent the majority of isoform events among the novel isoforms, with retained introns being the next most common event. The majority of alternative 5' ends correspond to genomic regions with features similar to those of the reference transcript 5' ends. However, a minority of alternative 5' ends showed markedly different features, including locations within protein-coding regions. Alternative 3' ends showed similar features to the reference transcript 3' ends, notably adenine-rich termination signals. Distinguishing features of retained introns could not be observed, except for a tendency towards shorter length and greater GC content compared with spliced introns. Expression of antisense and retained intron isoforms was detected at different intra-erythrocytic stages, suggesting developmental regulation of these isoform events. To gain insights into the possible functions of the novel isoforms, their protein-coding potential was assessed. Variants of P. falciparum proteins and novel proteins encoded by alternative open reading frames suggest that P. falciparum has a greater proteomic repertoire than the current annotation. We provide a catalog of annotated transcripts and encoded alternative proteins to support further studies on gene and protein regulation of this pathogen.

PharmVIP: A Web-Based Tool for Pharmacogenomic Variant Analysis and Interpretation.

The increasing availability of next generation sequencing (NGS) for personal genomics could promote pharmacogenomics (PGx) discovery and application. However, current tools for analysis and interpretation of pharmacogenomic variants from NGS data are inadequate, as none offer comprehensive analytic functions in a simple, web-based platform. In addition, no tools exist to analyze human leukocyte antigen (HLA) genes for determining potential risks of immune-mediated adverse drug reaction (IM-ADR). We describe PharmVIP, a web-based PGx tool, for one-stop comprehensive analysis and interpretation of genome-wide variants obtained from NGS platforms. PharmVIP comprises three main interpretation modules covering analyses of pharmacogenes involved in pharmacokinetics, pharmacodynamics and IM-ADR. The Guideline module provides Clinical Pharmacogenetics Implementation Consortium (CPIC) drug guideline recommendations based on the translation of genotypic data in genes having guidelines. The HLA module reports HLA genotypes, potential adverse drug reactions, and the relevant drug guidelines. The Pharmacogenes module is employed for prioritizing variants according to variant effect on gene function. Detailed, customizable reports are provided as exportable files and as an interactive web version. PharmVIP is a new integrated NGS workflow for the PGx community to facilitate discovery and clinical application.

Identifying transcript 5' capped ends in Plasmodium falciparum.

BACKGROUND: The genome of the human malaria parasite Plasmodium falciparum is poorly annotated, in particular, the 5' capped ends of its mRNA transcripts. New approaches are needed to fully catalog P. falciparum transcripts for understanding gene function and regulation in this organism. METHODS: We developed a transcriptomic method based on next-generation sequencing of complementary DNA (cDNA) enriched for full-length fragments using eIF4E, a 5' cap-binding protein, and an unenriched control. DNA sequencing adapter was added after enrichment of full-length cDNA using two different ligation protocols. From the mapped sequence reads, enrichment scores were calculated for all transcribed nucleotides and used to calculate P-values of 5' capped nucleotide enrichment. Sensitivity and accuracy were increased by combining P-values from replicate experiments. Data were obtained for P. falciparum ring, trophozoite and schizont stages of intra-erythrocytic development. RESULTS: 5' capped nucleotide signals were mapped to 17,961 non-overlapping P. falciparum genomic intervals. Analysis of the dominant 5' capped nucleotide in these genomic intervals revealed the presence of two groups with distinctive epigenetic features and sequence patterns. A total of 4,512 transcripts were annotated as 5' capped based on the correspondence of 5' end with 5' capped nucleotide annotated from full-length cDNA data. DISCUSSION: The presence of two groups of 5' capped nucleotides suggests that alternative mechanisms may exist for producing 5' capped transcript ends in P. falciparum. The 5' capped transcripts that are antisense, outside of, or partially overlapping coding regions may be important regulators of gene function in P. falciparum.

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

BACKGROUND: Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs. METHODS: We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors. RESULTS: A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5' and 3' untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants. DISCUSSION: The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.

Comparison of gene expression profiles between human erythroid cells derived from fetal liver and adult peripheral blood.

BACKGROUND: A key event in human development is the establishment of erythropoietic progenitors in the bone marrow, which is accompanied by a fetal-to-adult switch in hemoglobin expression. Understanding of this event could lead to medical application, notably treatment of sickle cell disease and ß-thalassemia. The changes in gene expression of erythropoietic progenitor cells as they migrate from the fetal liver and colonize the bone marrow are still rather poorly understood, as primary fetal liver (FL) tissues are difficult to obtain. METHODS: We obtained human FL tissue and adult peripheral blood (AB) samples from Thai subjects. Primary CD34+ cells were cultured in vitro in a fetal bovine serum-based culture medium. After 8 days of culture, erythroid cell populations were isolated by flow cytometry. Gene expression in the FL- and AB-derived cells was studied by Affymetrix microarray and reverse-transcription quantitative PCR. The microarray data were combined with that from a previous study of human FL and AB erythroid development, and meta-analysis was performed on the combined dataset. RESULTS: FL erythroid cells showed enhanced proliferation and elevated fetal hemoglobin relative to AB cells. A total of 1,391 fetal up-regulated and 329 adult up-regulated genes were identified from microarray data generated in this study. Five hundred ninety-nine fetal up-regulated and 284 adult up-regulated genes with reproducible patterns between this and a previous study were identified by meta-analysis of the combined dataset, which constitute a core set of genes differentially expressed between FL and AB erythroid cells. In addition to these core genes, 826 and 48 novel genes were identified only from data generated in this study to be FL up- and AB up-regulated, respectively. The in vivo relevance for some of these novel genes was demonstrated by pathway analysis, which showed novel genes functioning in pathways known to be important in proliferation and erythropoiesis, including the mitogen-activated protein kinase (MAPK) and the phosphatidyl inositol 3 kinase (PI3K)-Akt pathways. DISCUSSION: The genes with upregulated expression in FL cells, which include many novel genes identified from data generated in this study, suggest that cellular proliferation pathways are more active in the fetal stage. Erythroid progenitor cells may thus undergo a reprogramming during ontogenesis in which proliferation is modulated by changes in expression of key regulators, primarily MYC, and others including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), neuropilin and tolloid-like 2 (NETO2), branched chain amino acid transaminase 1 (BCAT1), tenascin XB (TNXB) and proto-oncogene, AP-1 transcription factor subunit (JUND). This reprogramming may thus be necessary for acquisition of the adult identity and switching of hemoglobin expression.

External Quality Assessment Scheme for HIV-1 Drug-Resistance Genotyping in Thailand.

The efficacy of antiretroviral (ARV) therapy can be compromised by the emergence and transmission of HIV-1 drug-resistant strains. HIV-1 drug-resistance (DR) genotypic testing thus plays an important role in the selection of optimal treatment regimens for HIV-infected individuals. Given the complexities of the testing procedures and the variety of approaches used, there is considerable potential for results to vary between laboratories. In Thailand, the national External Quality Assessment (EQA) scheme assesses the DR genotype testing performance of laboratories. Here, we evaluated the performance of laboratories in nucleotide sequencing and compared drug-resistance-associated mutations (DRMs) in the HIV-1 protease (PR) and reverse transcriptase (RT) genes during 2010-2015. The EQA samples in the 12 panels showed predominance for the CRF01_AE (85%) and subtype B (15%). Fourteen laboratory datasets were generated: eight using TruGene (TG), two using ViroSeq (VS), and four using in-house (IH) assays. All IH and VS laboratories had penalty scores <7, whereas five of the eight TG laboratories had fluctuating penalty scores. Moreover, seven and six TG laboratories could not amplify the two identical samples, 10B and 10E samples, or the CRF01_AE. Our findings demonstrate the requirement for laboratory participation in the ongoing EQA program and the optimization of kit assays using CRF01_AE samples. Our results also indicate that one advantage of participation is that the laboratories can monitor and investigate the source of laboratory errors.

The full-length genome characterization, genetic diversity and evolutionary analyses of Senecavirus A isolated in Thailand in 2016.

Senecavirus A (SVA) is a novel picornavirus that causes porcine idiopathic vesicular disease characterized by lameness, coronary band hyperemia, and vesicles on the snout and coronary bands. An increase in the detection rate of SVA in several countries suggests that the disease has become a widespread problem. Herein, we report the detection of SVA in Thailand and the characterization of full-length genomic sequences of six Thai SVA isolates. Phylogenetic, genetic, recombination, and evolutionary analyses were performed. The full-length genome, excluding the poly (A) tail of the Thai SVA isolates, was 7282 nucleotides long, with the genomic organization resembling other previously reported SVA isolates. Phylogenetic and genetic analyses based on full-length genome demonstrated that the Thai SVA isolates were grouped in a novel cluster, separated from SVA isolates from other countries. Although the Thai SVA isolates were closely related to 11-55910-3, the first SVA isolate from Canada, with 97.9-98.2%, but they are different. Evolutionary and recombinant analyses suggested that the Thai SVA isolates shared a common ancestor with the 11-55910-3 isolate. The positive selection in the VP4 and 3D genes suggests that the virus was not externally introduced, but rather continuously evolved in the population prior to the first detection. Addition, the presence of SVA could have been ignored due to the presence of other pathogens causing similar clinical diseases. This study warrants further investigations into molecular epidemiology and genetic evolution of the SVA in Thailand.

ToNER: A tool for identifying nucleotide enrichment signals in feature-enriched RNA-seq data.

BACKGROUND: Biochemical methods are available for enriching 5' ends of RNAs in prokaryotes, which are employed in the differential RNA-seq (dRNA-seq) and the more recent Cappable-seq protocols. Computational methods are needed to locate RNA 5' ends from these data by statistical analysis of the enrichment. Although statistical-based analysis methods have been developed for dRNA-seq, they may not be suitable for Cappable-seq data. The more efficient enrichment method employed in Cappable-seq compared with dRNA-seq could affect data distribution and thus algorithm performance. RESULTS: We present Transformation of Nucleotide Enrichment Ratios (ToNER), a tool for statistical modeling of enrichment from RNA-seq data obtained from enriched and unenriched libraries. The tool calculates nucleotide enrichment scores and determines the global transformation for fitting to the normal distribution using the Box-Cox procedure. From the transformed distribution, sites of significant enrichment are identified. To increase power of detection, meta-analysis across experimental replicates is offered. We tested the tool on Cappable-seq and dRNA-seq data for identifying Escherichia coli transcript 5' ends and compared the results with those from the TSSAR tool, which is designed for analyzing dRNA-seq data. When combining results across Cappable-seq replicates, ToNER detects more known transcript 5' ends than TSSAR. In general, the transcript 5' ends detected by ToNER but not TSSAR occur in regions which cannot be locally modeled by TSSAR. CONCLUSION: ToNER uses a simple yet robust statistical modeling approach, which can be used for detecting RNA 5'ends from Cappable-seq data, in particular when combining information from experimental replicates. The ToNER tool could potentially be applied for analyzing other RNA-seq datasets in which enrichment for other structural features of RNA is employed. The program is freely available for download at ToNER webpage (http://www4a.biotec.or.th/GI/tools/toner) and GitHub repository (https://github.com/PavitaKae/ToNER).

Evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in Thailand in 2008-2015.

Porcine epidemic diarrhea (PED) has been endemic causing sporadic outbreaks in Thailand since 2007. In 2014-2015, several herds had experienced severe PED outbreaks and the reason of the re-current outbreaks was unknown. Whether or not the introduction of exotic strains or continual evolution of existing PEDV, genetic analyses would provide a more understanding in its evolutionary pattern. In the study, 117 complete spike gene sequences of Thai PED virus (PEDV) collected from 2008 to 2015 were clustered along with 95 references of PEDV spike sequences, and analyzed with the US sequences dataset (n=99). The phylogenetic analysis demonstrated that Thai PEDV spike sequences were genetically diverse and had been influenced by multiple introduction of exotic strains. Although Thai PEDV have been evolved into 6 subgroups (TH1-6), Subgroup TH1 strains with the unique 9 nucleotides (CAA GGG AAT) insertion between 688th-689th position of spike (changing amino acid from N to TREY) insertion has become the dominant subgroup since 2014. Thai PEDV spike gene have higher evolutionary rate compare to that of the US sequences. One contributing factor would be the intra-recombination between subgroups. Thailand endemic strain should be assigned into new subclade of G2 (Thai pandemic variant).

The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thailand in 2015.

Porcine deltacoronavirus (PDCoV) was identified in intestinal samples collected from piglets with diarrhea in Thailand in 2015. Two Thai PDCoV isolates, P23_15_TT_1115 and P24_15_NT1_1215, were isolated and identified. The full-length genome sequences of the P23_15_TT_1115 and P24_15_NT1_1215 isolates were 25,404 and 25,407 nucleotides in length, respectively, which were relatively shorter than that of US and China PDCoV. The phylogenetic analysis based on the full-length genome demonstrated that Thai PDCoV isolates form a new cluster separated from US and China PDCoV but relatively were more closely related to China PDCoV than US isolates. The genetic analyses demonstrated that Thai PDCoVs have 97.0-97.8 and 92.2-94.0% similarities with China PDCoV at nucleotide and amino acid levels, respectively, but share 97.1-97.3 and 92.5-93.0 similarity with US PDCoV at the nucleotide and amino acid levels, respectively. Thai PDCoV possesses two discontinuous deletions of five amino acids in ORF1a/b region. One additional deletion of one amino acid was identified in P23_15_TT_1115. The variation analyses demonstrated that six regions (nt 1317-1436, 2997-3096, 19,737-19,836, 20,277-20,376, 21,177-21,276, and 22,371-22,416) in ORF1a/b and spike genes exhibit high sequence variation between Thai and other PDCoV. The analyses of amino acid changes suggested that they could potentially be from different lineages.

Full-length genome analysis of two genetically distinct variants of porcine epidemic diarrhea virus in Thailand.

Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007 and a pandemic variant containing an insertion and deletion in the spike gene was responsible for outbreaks. In 2014, there were further outbreaks of the disease occurring within four months of each other. In this study, the full-length genome sequences of two genetically distinct PEDV isolates from the outbreaks were characterized. The two PEDV isolates, CBR1/2014 and EAS1/2014, were 28,039 and 28,033 nucleotides in length and showed 96.2% and 93.6% similarities at nucleotide and amino acid levels respectively. In total, we have observed 1048 nucleotide substitutions throughout the genome. Compared to EAS1/2014, CBR1/2014 has 2 insertions of 4 ((56)GENQ(59)) and 1 ((140)N) amino acid positions 56-59 and 140, and 2 deletions of 2 ((160)DG(161)) and 1 ((1199)Y) amino acid positions 160-161 and 1199. The phylogenetic analysis based on full-length genome of CBR1/2014 isolate has grouped the virus with the pandemic variants. In contrast, EAS1/2014 isolate was grouped with CV777, LZC and SM98, a classical variant. Our findings demonstrated the emergence of EAS1/2014, a classical variant which is novel to Thailand and genetically distinct from the currently circulating endemic variants. This study warrants further investigations into molecular epidemiology and genetic evolution of the PEDV in Thailand.

Estimating mRNA lengths from Plasmodium falciparum genes by Virtual Northern RNA-seq analysis.

Accurate gene models are essential for understanding parasite biology. However, transcript structure information is lacking for most parasite genes. Here, we describe "Virtual Northern" analysis of the malaria parasite Plasmodium falciparum to address this issue. RNA-seq libraries were made from size-fractionated RNA. Transcript sizes for 3052 genes were inferred from the read counts in each library. The data show that for almost half of the transcripts, the combined untranslated regions are more than twice the length of the open reading frame. Furthermore, we identified novel polycistronic, or gene overlapping, transcripts that suggest revisions to current gene models are needed.

Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study.

BACKGROUND: Control of malaria is threatened by emerging parasite resistance to artemisinin and derivative drug (ART) therapies. The molecular detail of how Plasmodium malaria parasites respond to ART and how this could contribute to resistance are not well understood. To address this question, we performed a transcriptomic study of dihydroartemisinin (DHA) response in P. falciparum K1 strain and in P. berghei ANKA strain using microarray and RNA-seq technology. RESULTS: Microarray data from DHA-treated P. falciparum trophozoite stage parasites revealed a response pattern that is overall less trophozoite-like and more like the other stages of asexual development. A meta-analysis of these data with previously published data from other ART treatments revealed a set of common differentially expressed genes. Notably, ribosomal protein genes are down-regulated in response to ART. A similar pattern of trophozoite transcriptomic change was observed from RNA-seq data. RNA-seq data from DHA-treated P. falciparum rings reveal a more muted response, although there is considerable overlap of differentially expressed genes with DHA-treated trophozoites. No genes are differentially expressed in DHA-treated P. falciparum schizonts. The transcriptional response of P. berghei to DHA treatment in vivo in infected mice is similar to the P. falciparum in vitro culture ring and trophozoite responses, in which ribosomal protein genes are notably down-regulated. CONCLUSIONS: Ring and trophozoite stage Plasmodium respond to ART by arresting metabolic processes such as protein synthesis and glycolysis. This response can be protective in rings, as shown by the phenomenon of dormancy. In contrast, this response is not as protective in trophozoites owing to their commitment to a highly active and vulnerable metabolic state. The lower metabolic demands of schizonts could explain why they are less sensitive and unresponsive to ART. The ART response pattern is revealed clearly from RNA-seq data, suggesting that this technology is of great utility for studying drug response in Plasmodium.