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RNA sequencing

Anastasia K Atabekova, Anna V Pankratenko, Svetlana S Makarova, Ekaterina A Lazareva, Robert A Owens, Andrey G Solovyev, Sergey Y Morozov
Human B-cell receptor-associated protein BAP31 (HsBAP31) is the endoplasmic reticulum-resident protein involved in protein sorting and transport as well as pro-apoptotic signaling. Plant orthologs of HsBAP31 termed 'plant BAP-like proteins' (PBL proteins) have thus far remained unstudied. Recently, the PBL protein from Nicotiana tabacum (NtPBL) was identified as an interactor of Nt-4/1, a plant protein known to interact with plant virus movement proteins and affect the long-distance transport of potato spindle tuber viroid (PSTVd) via the phloem...
October 19, 2016: Biochimie
L C Perkin, E N Elpidina, B Oppert
Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double-stranded RNA (dsRNA) constructs of different regions of the gene (3', middle, 5' and entire coding regions). RNA sequencing and quantitation (RNA-seq) was used to evaluate knockdown and specificity amongst the treatments...
October 22, 2016: Insect Molecular Biology
Zheng Fang, Jingxu Shao, Qingbei Weng
Cells of the P8-Se301-C1 strain are Spodoptera exigua cell clones that each harbor a partial version of the S. exigua multiple nucleopolyhedrovirus (SeMNPV) genome and which are resistant to homologous SeMNPV infections. The cells produce no viral progeny, suggesting that the infection is a latent-like viral infection. To investigate the SeMNPV genes harbored in the P8-Se301-C1 cells, the de novo transcriptomes of P8-Se301-C1 cells and S. exigua Se301 cells were analyzed and compared. A total of 54,569,296 reads were obtained from the P8-Se301-C1 cells that yielded 112,565 final unigenes with a mean length of 1,093 nt...
October 18, 2016: Virologica Sinica
Slava Ilnytskyy, Andriy Bilichak
Next-generation sequencing became a method of choice for the investigation of small RNA transcriptomes in plants and animals. Although a technical side of sequencing itself is becoming routine, and experimental costs are affordable, data analysis still remains a challenge, especially for researchers with limited computational experience. Here, we present a detailed description of a computational workflow designed to take raw sequencing reads as input, to obtain small RNA predictions, and to detect the differentially expressed microRNAs as a result...
2017: Methods in Molecular Biology
Andriy Bilichak, Andrey Golubov, Igor Kovalchuk
The discovery of small RNAs in plants and animals almost two decades ago attracted a significant interest towards epigenetic regulation of gene expression and the practical implementation of the gained knowledge in applied studies. New and sometimes unexpected functions have been ascribed to sRNAs almost every couple of years since their discovery, hence indicating that the complete role of sRNAs in plant and animal physiology is still barely understood. Next-generation sequencing technologies allow to generate high-resolution profiles of sRNAs for the consequent analysis and possibly to discover novel functions of sRNAs...
2017: Methods in Molecular Biology
Masayuki Tsuzuki, Yuichiro Watanabe
Small RNAs are key molecules in RNA silencing pathways that exert the sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs), trans-acting short interfering RNAs (tasiRNAs), and heterochromatic siRNAs (hc-siRNAs), play an important role in switching or orchestrating biological processes during the development and at the onset of stress responses. These endogenous and exogenous small RNAs are mainly 20-24 nucleotides in length...
2017: Methods in Molecular Biology
Martine Boccara, Alexis Sarazin, Bernard Billoud, Agnes Bulski, Louise Chapell, David Baulcombe, Vincent Colot
Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals...
2017: Methods in Molecular Biology
Sara Hernández-Castellano, Geovanny I Nic-Can, Clelia De-la-Peña
Among the epigenetic mechanisms studied with a greater interest in the last decade are the microRNAs (miRNAs). These small noncoding RNA sequences that are approximately 17-22 nucleotides in length play an essential role in many biological processes of various organisms, including plants. The analysis of spatiotemporal expression of miRNAs provides a better understanding of the role of these small molecules in plant development, cell differentiation, and other processes; but such analysis is also an important method for the validation of biological functions...
2017: Methods in Molecular Biology
Pei Ma, Haitao Wang, Lu Han, Wei Jing, Xin Zhou, Zhisu Liu
Small nucleolar RNAs (snoRNAs) as a novel molecular species may have significant and comprehensive influences on the development and progression of hepatocellular carcinoma (HCC). We recently characterized snoRNA transcriptome signatures in HCC tissues by small RNA sequencing and found that small nucleolar RNA 78 (SNORD78) was associated with HCC. However, little is known about the pathological role of SNORD78 in HCC patients. This study aimed to profile SNORD78 expression signature and then to explore the pathogenesis of SNORD78 in HCC...
October 21, 2016: Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine
Liuyu Wu, Xiao Xiong, Xuming Wang, Tianrong Xin, Jing Wang, Zhiwen Zou, Bin Xia
The bluish yellow lappet moth, Trabala vishnou guttata is an extraordinarily important pest in China. The complete mitochondrial genome is sequenced and determined firstly, which is based on traditional PCR amplification and primer walking methods with a length of 15,281 bp, including 13 protein-coding (PCG) genes, 22 transfer RNA (rRNA) genes, two ribosomal RNA (tRNA) genes, and an A + T-rich region. The gene order and orientation of the T. vishnou guttata mitogenome were identical to the other sequenced Lasiocampidae species...
October 21, 2016: Genetica
Liyan Song, Shu Yang, Hongjie Liu, Jing Xu
Little is known regarding how bacterial communities assemble at landfill, as well as how the environment shapes the composition of bacterial community. In this study, up to 42 refuse samples from a large-scale landfill in China were physicochemically and phylogenetically investigated. 16S ribosomal RNA (rRNA) gene-based Illumina MiSeq sequencing (nine samples) revealed that representatives of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and Bacteroidetes were dominant in the refuse samples, which was similar to a previous study on landfill leachate by using 454 pyrosequencing...
October 21, 2016: Applied Microbiology and Biotechnology
Maoqi Xu, Liang Chen
The individual sample heterogeneity is one of the biggest obstacles in biomarker identification for complex diseases such as cancers. Current statistical models to identify differentially expressed genes between disease and control groups often overlook the substantial human sample heterogeneity. Meanwhile, traditional nonparametric tests lose detailed data information and sacrifice the analysis power, although they are distribution free and robust to heterogeneity. Here, we propose an empirical likelihood ratio test with a mean-variance relationship constraint (ELTSeq) for the differential expression analysis of RNA sequencing (RNA-seq)...
October 21, 2016: Briefings in Bioinformatics
Dennis Walczyk, Markus Gößringer, Walter Rossmanith, Timofei S Zatsepin, Tatiana S Oretskaya, Roland K Hartmann
Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino or 2'-O-methyl substitutions...
October 18, 2016: Journal of Molecular Biology
Ryo Nakao, Keita Matsuno, Yongjin Qiu, Junki Maruyama, Nao Eguchi, Naganori Nao, Masahiro Kajihara, Kentaro Yoshii, Hirofumi Sawa, Ayato Takada, Chihiro Sugimoto
Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis...
October 15, 2016: Ticks and Tick-borne Diseases
Sergi Sayols, Denise Scherzinger, Holger Klein
BACKGROUND: PCR clonal artefacts originating from NGS library preparation can affect both genomic as well as RNA-Seq applications when protocols are pushed to their limits. In RNA-Seq however the artifactual reads are not easy to tell apart from normal read duplication due to natural over-sequencing of highly expressed genes. Especially when working with little input material or single cells assessing the fraction of duplicate reads is an important quality control step for NGS data sets...
October 21, 2016: BMC Bioinformatics
Binyi Lin, Tianchi Chen, Qijun Zhang, Xiaoxiao Lu, Zhiyun Zheng, Jun Ding, Jinfeng Liu, Zhe Yang, Lei Geng, Liming Wu, Lin Zhou, Shusen Zheng
To investigate the potential oncogene promoting recurrence of hepatocellular carcinoma (HCC) following liver transplantation (LT), throughput RNA sequencing was performed in a subgroup of HCC patients. The up-regulated FAM83D in HCC tissues was found and further verified in 150 patients by real-time PCR and immunohistochemistry. FAM83D overexpression significantly correlated with high HCC recurrence rate following LT and poor HCC characteristics such as high AFP, poor differentiation. Of cancer stem cells (CSCs) markers, CD44 expression was effectively suppressed when FAM83D was knocked down by siRNA...
October 18, 2016: Oncotarget
Na Li, Yunhuan Yan, Angke Zhang, Jiming Gao, Chong Zhang, Xue Wang, Gaopeng Hou, Gaiping Zhang, Jinbu Jia, En-Min Zhou, Shuqi Xiao
Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification...
October 17, 2016: Oncotarget
Anna Chung-Kwan Tse, Jing-Woei Li, Simon Yuan Wang, Ting-Fung Chan, Keng Po Lai, Rudolf Shiu-Sun Wu
Hypoxia is a global environmental concern and poses a significant threat to aquatic ecosystems, including the sustainability of natural fish populations. The deleterious effects of hypoxia on fish reproductive fitness, as mediated by disruption of sex hormones and gene expression along the Brain-Pituitary-Gonad axis, have been well documented. Recently, we further demonstrated that the observed disruption of steroidogenesis in the ovary of marine medaka Oryzias melastigma is mediated through microRNAs (miRNAs)...
October 8, 2016: Aquatic Toxicology
Kyung-Ha Lee, Peipei Zhang, Hong Joo Kim, Diana M Mitrea, Mohona Sarkar, Brian D Freibaum, Jaclyn Cika, Maura Coughlin, James Messing, Amandine Molliex, Brian A Maxwell, Nam Chul Kim, Jamshid Temirov, Jennifer Moore, Regina-Maria Kolaitis, Timothy I Shaw, Bing Bai, Junmin Peng, Richard W Kriwacki, J Paul Taylor
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles...
October 20, 2016: Cell
Hélène Quach, Maxime Rotival, Julien Pothlichet, Yong-Hwee Eddie Loh, Michael Dannemann, Nora Zidane, Guillaume Laval, Etienne Patin, Christine Harmant, Marie Lopez, Matthieu Deschamps, Nadia Naffakh, Darragh Duffy, Anja Coen, Geert Leroux-Roels, Frederic Clément, Anne Boland, Jean-François Deleuze, Janet Kelso, Matthew L Albert, Lluis Quintana-Murci
Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli-ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus-and mapped expression quantitative trait loci (eQTLs)...
October 20, 2016: Cell
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