RnaseIII and T4 Polynucleotide Kinase Sequence Biases and Solutions During RNA-Seq Library Construction

Background: RNA-seq is a next generation sequencing method with a wide range of applications including single nucleotide polymorphism (SNP) detection, splice junction identification, and gene expression level measurement. However, the RNA-seq sequence data can be biased during library constructions resulting in incorrect data for SNP, splice junction, and gene expression studies. Here, we developed new library preparation methods to limit such biases. Results: A whole transcriptome library prepared for the SOLiD system displayed numerous read duplications (pile-ups) and gaps in known exons. The pile-ups and gaps of the whole transcriptome library caused a loss of SNP and splice junction information and reduced the quality of gene expression results. Further, we found clear sequence biases for both 5' and 3' end reads in the whole transcriptome library. To remove this bias, RNaseIII fragmentation was replaced with heat fragmentation. For adaptor ligation, T4 Polynucleotide Kinase (T4PNK) was used following heat fragmentation. However, its kinase and phosphatase activities introduced additional sequence biases. To minimize them, we used OptiKinase before T4PNK. Our study further revealed the specific target sequences of RNaseIII and T4PNK. Conclusions: Our results suggest that the heat fragmentation removed the RNaseIII sequence bias and significantly reduced the pile-ups and gaps. OptiKinase minimized the T4PNK sequence biases and removed most of the remaining pile-ups and gaps, thus maximizing the quality of RNA-seq data.National Institute on Alcohol Abuse and Alcoholism (NIAAA) AA12404, AA019382, AA020926, AA016648National Institutes of Health (NIH) R01 GM088344Waggoner Center for Alcohol and Addiction Researc

On-pump coronary artery bypass graft surgery: biochemical, hormonal and cellular features

OBJECTIVE: The authors sought to assess biochemical, hormonal and cellular repercussions from use of cardiopulmonary bypass (CPB) in coronary artery bypass graft (CABG) surgery. METHODS: Eighteen patients underwent on-pump CABG surgery. Mean time of CPB was 80.3 minutes. Hormonal, biochemical and cellular measurements were taken in some time points - preoperatively, immediately after coming off CPB, 24 and 48 hours postoperatively. Friedman and Wilcoxon tests were applied based on significance level of 5%. RESULTS: There was activation and significant elevation of total leukocytes and neutrophils count over CPB, remaining this way up to 48 hours postoperatively. Total platelets count, in turn, was marked by relevant reduction immediately after coming off CPB as well as in two postoperative time points. Serum levels of total proteins and albumin, immediately after coming off CPB and also in two postoperative time points, were significantly decreased comparing with preoperative status. There was remarkable reduction of total T3, free T3 and total T4 particularly up to first 24 hours postoperatively. CONCLUSION: In on-pump CABG surgery, inflammatory effects encompass activation of total leukocytes, neutrophils and platelets, reduction of serum level of total proteins and albumin and decreased thyroid hormones levels, especially within first postoperative 24 hours.OBJETIVO: Avaliar repercussões bioquímicas, hormonais e celulares decorrentes do emprego de circulação extracorpórea (CEC) em cirurgia de revascularização miocárdica. MÉTODOS: Dezoito pacientes foram submetidos à cirurgia de revascularização miocárdica com emprego de CEC. A duração média da CEC foi de 80,3 minutos. Dosagens hormonais, bioquímicas e celulares foram realizadas nos seguintes tempos: pré-operatório, logo após a saída de CEC, 24 horas e 48 horas de pós-operatório. Os testes de Friedman e Wilcoxon foram aplicados, considerando-se o nível de significância 5%. RESULTADOS: Houve ativação e elevação significante do número de leucócitos totais e neutrófilos durante o período de CEC, de tal forma que esta condição foi detectada logo após a saída de CEC, mantendo-se assim até 48 horas de pós-operatório. O número total de plaquetas, por sua vez, caracterizou-se por decréscimo relevante logo após a saída de CEC, como também nos dois momentos pós-operatórios de observação. A concentração sérica de proteínas totais e albumina, logo após a saída de CEC e nos dois momentos pós-operatórios de observação, foi significativamente menor em relação aos níveis encontrados no período pré-operatório. Houve decréscimo acentuado dos níveis séricos de T3 total e T3 livre, sobretudo até as primeiras 24 horas de pós-operatório. De forma análoga, notou-se padrão semelhante quanto aos níveis séricos de T4 total. CONCLUSÃO: Em cirurgias de revascularização miocárdica, os efeitos inflamatórios da CEC compreendem ativação de leucócitos, neutrófilos e plaquetas, redução na concentração sérica de proteínas totais e albumina e decréscimo dos níveis séricos de hormônios tireoidianos, sobretudo, nas primeiras 24 horas de pós-operatório.Universidade de São Paulo Faculdade de MedicinaUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Genome-Wide Profiling of Circular RNAs in the Rapidly Growing Shoots of Moso Bamboo (Phyllostachys edulis).

Circular RNAs, including circular exonic RNAs (circRNA), circular intronic RNAs (ciRNA) and exon-intron circRNAs (EIciRNAs), are a new type of noncoding RNAs. Growing shoots of moso bamboo (Phyllostachys edulis) represent an excellent model of fast growth and their circular RNAs have not been studied yet. To understand the potential regulation of circular RNAs, we systematically characterized circular RNAs from eight different developmental stages of rapidly growing shoots. Here, we identified 895 circular RNAs including a subset of mutually inclusive circRNA. These circular RNAs were generated from 759 corresponding parental coding genes involved in cellulose, hemicellulose and lignin biosynthetic process. Gene co-expression analysis revealed that hub genes, such as DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), MAINTENANCE OF METHYLATION (MOM), dicer-like 3 (DCL3) and ARGONAUTE 1 (AGO1), were significantly enriched giving rise to circular RNAs. The expression level of these circular RNAs presented correlation with its linear counterpart according to transcriptome sequencing. Further protoplast transformation experiments indicated that overexpressing circ-bHLH93 generating from transcription factor decreased its linear transcript. Finally, the expression profiles suggested that circular RNAs may have interplay with miRNAs to regulate their cognate linear mRNAs, which was further supported by overexpressing miRNA156 decreasing the transcript of circ-TRF-1 and linear transcripts of TRF-1. Taken together, the overall profile of circular RNAs provided new insight into an unexplored category of long noncoding RNA regulation in moso bamboo

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Texas Student Medi

BlogForever D5.3: User Questionnaires and Reports

This report presents the feedback gathered from third party users during the BlogForever Case Studies. Therefore, the research framework is defined and the case studies results are presented, followed by a summary of conclusions and remarks