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

RNA-seq data analysis from DHA treatment of P. falciparum Limma results from 1 h treatments with 500 nM DHA in P. falciparum K1 rings, trophozoites and schizonts. (XLS 2040 kb

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 β-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

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 significantly enriched nucleotides detected by ToNER and TSSAR.

<p>Venn diagrams show overlaps of nucleotides detected as significantly enriched (p<0.05) from ToNER and TSSAR analyses for replicate 1 (cappable1), replicate 2 (cappable2), and the Fisher’s combined results from the two replicates (combine). The number of whole transcriptome significantly enriched positions are shown in black, whereas the number of enriched positions corresponding to known transcription start sites annotated in RegulonDB are shown in red.</p

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

<div><p>Background</p><p>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.</p><p>Results</p><p>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 <i>Escherichia coli</i> 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.</p><p>Conclusion</p><p>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 (<a href="http://www4a.biotec.or.th/GI/tools/toner" target="_blank">http://www4a.biotec.or.th/GI/tools/toner</a>) and GitHub repository (<a href="https://github.com/PavitaKae/ToNER" target="_blank">https://github.com/PavitaKae/ToNER</a>).</p></div

Cappable-seq data analysis results for ToNER and TSSAR.

<p>Cappable-seq data analysis results for ToNER and TSSAR.</p

The normal Q-Q plots of the calculated enrichment scores from Cappable-seq data.

<p>Q-Q plots of enrichment score quantiles calculated from Cappable-seq data (vertical axes) versus normally distributed theoretical quantiles (horizontal axes) are shown for scores before and after Box-Cox transformation for experimental replicate 1 (A) and replicate 2 (B). The critical value of enrichment score cutoff at p = 0.05 is indicated by the green horizontal line. The R² linear correlation coefficients are also shown on the plots. The Box-Cox lambda values used for transformation of enrichment scores are 0.1033 and 0.1136 for replicate 1 and 2 respectively.</p

Example of an annotated TSS which is located in an unmodeled region by TSSAR in both Cappable-seq and dRNA-seq datasets.

<p>The plots of normalized read depth values of enriched and unenriched libraries including the corresponding enrichment scores reported by ToNER are shown for the 100 bp window (from -50 bp upstream to +50 bp downstream) of an annotated TSS position of <i>E</i>. <i>coli</i> (NC_000913.2 position 3,309,808; ‘-‘ strand). Data from the Cappable-seq protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178483#pone.0178483.ref004" target="_blank">4</a>] are shown for Cappable-seq replicate 1 (A) and Cappable-seq replicate 2 (B). Data from the dRNA-seq protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178483#pone.0178483.ref013" target="_blank">13</a>] are shown for dataset M63_0.4_B1_L1_GA (C) and dataset M63_0.4_B2_L1_HS2 (D). The ToNER calculated p-values of the annotated TSS position reported in Cappable-seq replicate 1, replicate 2, and combined result are 0.0043, 0.0126, and 0.0017, respectively. For dRNA-seq, the p-values reported in replicate B1, replicate B2, and combined result are 0.0549, 0.1046, and 0.0125, respectively.</p

The workflow of the ToNER software.

<p>The workflow of the ToNER software.</p