Deep sequencing reveals complex mechanisms of microRNA regulation during retinoic acid-induced neuronal differentiation of mesenchymal stem cells.

Retinoic acid (RA) has an important role in nervous system development; exogenous RA could induce stem cells towards neural lineage cells. However, the miRNA regulation mechanism and biological process of this induction require further exploration. In this study, using high-throughput sequencing results, we evaluated the microRNA profiles of neurally differentiated adipose-derived mesenchymal stem cells (ASCs), summarized several crucial microRNAs that profoundly contributed to the differentiation process, and speculated that several miRNAs were likely to mimic RA or other factors to induce the neuronal differentiation of stem cells. The GO terms and KEGG PATHWAY in the DAVID tool were used to elucidate the biological process of RA induction. Finally, we described a network for clarifying the relationship among the miRNAs, target genes and signaling pathways. These findings will be beneficial for understanding the induction mechanism and supporting the application of RA in stem cell transformation.

Small RNA and degradome sequencing reveals important microRNA function in Astragalus chrysochlorus response to selenium stimuli.

Selenium (Se), an essential element, plays important roles in human health as well as environmental sustainability. Se hyperaccumulating plants are thought as an alternative selenium resource, recently. Astragalus species are known as hyperaccumulator of Se by converting it to nonaminoacid compounds. However, Se-metabolism-related hyperaccumulation is not elucidated in plants yet. MicroRNAs (miRNAs) are key molecules in many biological and metabolic processes via targeting mRNAs, which may also play an important role in Se accumulation in plants. In this study, we identified 418 known miRNAs, belonging to 380 families, and 151 novel miRNAs induced by Se exposure in Astragalus chyrsochlorus callus. Among known miRNAs, the expression of 287 families was common in both libraries, besides 71 families were expressed only in Se-treated sample, whereas 60 conserved families were expressed in control tissue. miR1507a, miR1869 and miR2867-3p were mostly up-regulated, whereas miR1507-5p and miR8781b were significantly down-regulated by Se exposure. Computational analysis shows that the targets of miRNAs are involved in different types of biological mechanisms including 47 types of cellular component, 103 types of molecular function and 144 types of biological process. Degradome analysis shows that 1256 mRNAs were targeted by 499 miRNAs. We conclude that some known and novel miRNAs such as miR167a, miR319, miR1507a, miR4346, miR7767-3p, miR7800, miR9748 and miR-n93 target transcription factors, disease resistance proteins and some specific genes like cysteine synthase and might be related to plant hormone signal transduction, plant-pathogen interaction and sulphur metabolism pathways.

Comparative Analysis of Salt Responsive MicroRNAs in Two Sweetpotato [Ipomoea batatas (L.) Lam.] Cultivars With Different Salt Stress Resistance.

Sweetpotato [Ipomoea batatas (L.) Lam.] is an important food, vegetable and economic crop, but its productivity is remarkably affected by soil salinity. MiRNAs are a class of endogenous non-coding small RNAs that play an important role in plant resistance to salt stress. However, the function of miRNAs still remains largely unknown in sweetpotato under salt stress. Previously, we identified salt-responsive miRNAs in one salt-sensitive sweetpotato cultivar "Xushu 32." In this study, we identified miRNAs in another salt-tolerant cultivar "Xushu 22" by high-throughput deep sequencing and compared the salt-responsive miRNAs between these two cultivars with different salt sensitivity. We identified 687 miRNAs in "Xushu 22," including 514 known miRNAs and 173 novel miRNAs. Among the 759 miRNAs from the two cultivars, 72 and 109 miRNAs were specifically expressed in "Xushu 32" and "Xushu 22," respectively, and 578 miRNAs were co-expressed. The comparison of "Xushu 32" and "Xushu 22" genotypes showed a total of 235 miRNAs with obvious differential expression and 177 salt-responsive miRNAs that were obviously differently expressed between "Xushu 32" and "Xushu 22" under salt stress. The target genes of the miRNAs were predicted and identified using the Target Finder tool and degradome sequencing. The results showed that most of the targets were transcription factors and proteins related to metabolism and stress response. Gene Ontology analysis revealed that these target genes are involved in key pathways related to salt stress response and secondary redox metabolism. The comparative analysis of salt-responsive miRNAs in sweetpotato cultivars with different salt sensitivity is helpful for understanding the regulatory pattern of miRNA in different sweetpotato genotypes and improving the agronomic traits of sweetpotato by miRNA manipulation in the future.

Small RNA and Degradome Sequencing Reveal Important MicroRNA Function in Nicotiana tabacum Response to Bemisia tabaci.

MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, are key molecules in many biological and metabolic processes of plant growth, development and stress response via targeting mRNAs. The phloem-feeding insect whitefly Bemisia tabaci (Hemiptera, Aleyrodidae) is a serious pest that causes devastating harm to agricultural production worldwide. However, the function of host miRNAs in the response to whitefly infestation remains unclear. Here, we sequenced the small RNA and degradome of tobacco (Nicotiana tabacum L.), after and before infestation by B. tabaci. We identified 1291 miRNAs belonging to 138 miRNA families including 706 known miRNAs and 585 novel miRNAs. A total of 47 miRNAs were differentially expressed, of which 30 were upregulated and 17 were downregulated by whitefly exposure. Then, computational analysis showed that the target genes of differential miRNAs were involved in R gene regulation, plant innate immunity, plant pathogen defense, the plant hormone signal pathway and abiotic stress tolerance. Furthermore, degradome analysis demonstrated that 253 mRNAs were cleaved by 66 miRNAs. Among them, the targets cleaved by upregulated miR6025, miR160, miR171, miR166 and miR168 are consistent with our prediction, suggesting that pathogen-related miRNAs may function in plant defense against whitefly. Moreover, our results show that plant miRNA response and miRNA-mediated post-transcriptional regulation for phloem-feeding insect infestation are similar to pathogen invasion. Our study provides additional data to further elucidate how host plants respond and defend the phloem-feeding insects.

Assessing Genome-Wide Diversity in European Hantaviruses through Sequence Capture from Natural Host Samples.

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

Workup of Human Blood Samples for Deep Sequencing of HIV-1 Genomes.

We describe a detailed protocol for the manual workup of blood (plasma/serum) samples from individuals infected with the human immunodeficiency virus type 1 (HIV-1) for deep sequence analysis of the viral genome. The study optimizing the assay was performed in the context of the BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project, which analyzes complete viral genomes from more than 3000 HIV-1-infected Europeans with high-throughput deep sequencing techniques. The goal of the BEEHIVE project is to determine the contribution of viral genetics to virulence. Recently we performed a pilot experiment with 125 patient plasma samples to identify the method that is most suitable for isolation of HIV-1 viral RNA for subsequent long-amplicon deep sequencing. We reported that manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) provides superior results over robotically extracted RNA. The latter approach used the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics), the QIAcube robotic system (Qiagen), or the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular). Here we present a detailed protocol for the labor-intensive manual extraction method that yielded the best results.

Whole genome deep sequencing revealed host impact on population structure, variation and evolution of Rice stripe virus.

High-throughput deep sequencing and variant detection showed that variations of Rice stripe virus (RSV) populations obtained from small brown planthopper-transmitted rice plants and sap-inoculated N. benthamiana plants were single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels). The SNPs were more uniform across RSV genome, but InDels occurred mainly in the intergenic regions (IRs) and in the 5' or 3' noncoding regions. There were no clear patterns of InDels, although the inserted sequences were all from virus itself. Six, one, and one non-synonymous substitutions were respectively observed in the RdRP ORF, IR and the movement protein ORF. These non-synonymous substitutions were found to be stable, resulting in new consensus sequences in the NBL11 RSV population. Furthermore, the numbers of SNPs and InDels in RSV genome from N. benthamiana plants were much higher than that from O. sativa plants. These differences are likely caused by selection pressures generated by different host plants.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing.

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Screening on the differentially expressed miRNAs in zebrafish (Danio rerio) exposed to trace ß-diketone antibiotics and their related functions.

The toxicity of ß-diketone antibiotics (DKAs) to larval and adult zebrafish (Danio rerio) was investigated by miRNA sequencing and bioinformatics analyses. In control and DKA-exposed groups, 215 differentially expressed miRNAs were screened, and 4076 differential target genes were predicted. Among 51 co-differentially expressed genes, 45 were annotated in KOG functional classification, and 34 in KEGG pathway analysis. The homology analysis of 20 miRNAs with human hsa-miRNAs demonstrated 17 high homologous sequences. The expression levels of 12 miRNAs by qRT-PCR were consistent with those by sRNA-seq. A regulatory network for 4 positive miRNA genes (dre-miR-10, -96, -92 and -184) was plotted, and the high-degree of connectivity between miRNA-gene pairs suggests that these miRNAs play critical roles during zebrafish development. The consistent expression of dre-miR-184 and dre-miR-96 was proved in 120-hpf zebrafish brain, gill, otoliths and lateral line neuromast by qRT-PCR, miRNA-seq, W-ISH and ISH. DKA-exposure led to vacuolation of interstitial cells, reduced number of neurons, glial cell proliferation and formation of glial scar, and the obvious abnormality of cell structure might result from abnormal expression of differentially expressed miRNAs. In general, chronic DKA-exposure resulted in comprehensively toxic effects on larval and adult zebrafish tissues, especially for nervous system.

Peripheral blood transcriptome sequencing reveals rejection-relevant genes in long-term heart transplantation.

BACKGROUND: Peripheral blood-based gene expression patterns have been investigated as biomarkers to monitor the immune system and rule out rejection after heart transplantation. Recent advances in the high-throughput deep sequencing (HTS) technologies provide new leads in transcriptome analysis. METHODS: By performing Solexa/Illumina's digital gene expression (DGE) profiling, we analyzed gene expression profiles of PBMCs from 6 quiescent (grade 0) and 6 rejection (grade 2R&3R) heart transplant recipients at more than 6 months after transplantation. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was carried out in an independent validation cohort of 47 individuals from three rejection groups (ISHLT, grade 0,1R, 2R&3R). RESULTS: Through DGE sequencing and qPCR validation, 10 genes were identified as informative genes for detection of cardiac transplant rejection. A further clustering analysis showed that the 10 genes were not only effective for distinguishing patients with acute cardiac allograft rejection, but also informative for discriminating patients with renal allograft rejection based on both blood and biopsy samples. Moreover, PPI network analysis revealed that the 10 genes were connected to each other within a short interaction distance. CONCLUSIONS: We proposed a 10-gene signature for heart transplant patients at high-risk of developing severe rejection, which was found to be effective as well in other organ transplant. Moreover, we supposed that these genes function systematically as biomarkers in long-time allograft rejection. Further validation in broad transplant population would be required before the non-invasive biomarkers can be generally utilized to predict the risk of transplant rejection.