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8th ACM International Conference on Bioinformatics, Computational Biology,and Health Informatics (ACM-BCB)

作者： Ju, Chelsea J. -T. Li, Ruirui Wu, Zhengliang Jiang, Jyun-Yu Yang, Zhao Wang, Wei Univ Calif Los Angeles Dept Comp Sci Los Angeles CA 90024 USA

ISBN: (纸本)9781450347228

The advent of RNA-Seq has made it possible to quantify transcript expression on a large scale simultaneously. This technology generates small fragments of each transcript sequence, known as sequencing reads. As the first step of data analysis towards expression quantification, most of the existing methods align these reads to a reference genome or transcriptome to establish their origins. However, read alignment is computationally costly. Recently, a series of methods have been proposed to perform a lightweight quantification analysis in an alignment-free manner. These methods utilize the notion of k-mers, which are short consecutive sequences representing the signatures of each transcript, to estimate the relative abundance from RNA-Seq reads. Current k-mer based approaches make use of a set of fixed size k-mers;however, the true signatures of each transcript may not exist in a fixed size. In this paper, we demonstrate the importance of k-mers selection in transcript abundance estimation. We propose a novel method, Fleximer, to efficiently discover and select an optimal set of k-mers with flexible lengths. Using both simulated and real datasets, we show that, with fewer k-mers, Fleximer is able to cover the similar amount of reads as Sailfish and Kallisto. The selected k-mers own more distinguishing features, and thus substantially reduce the errors in transcript abundance estimation.

关键词： RNA-Seq Suffix tree Aho-Corasick algorithm EM algorithm transcript quantification

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Web-based bioinformatics workflows for end-to-end RNA-seq data computation and analysis in agricultural animal species

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BMC GENOMICS 2016年第1期17卷 1-7页

作者： Li, Weizhong Richter, R. Alexander Jung, Yunsup Zhu, Qiyun Li, Robert W. J Craig Venter Inst La Jolla CA 92037 USA USDA ARS Anim Genom & Improvement Lab Beltsville MD 20705 USA

Background: Remarkable advances in Next Generation Sequencing (NGS) technologies, bioinformatics algorithms and computational technologies have significantly accelerated genomic research. However, complicated NGS data analysis still remains as a major bottleneck. RNA-seq, as one of the major area in the NGS field, also confronts great challenges in data analysis. Results: To address the challenges in RNA-seq data analysis, we developed a web portal that offers three integrated workflows that can perform end-to-end compute and analysis, including sequence quality control, read-mapping, transcriptome assembly, reconstruction and quantification, and differential analysis. The first workflow utilizes Tuxedo (Tophat, Cufflink, Cuffmerge and Cuffdiff suite of tools). The second workflow deploys Trinity for de novo assembly and uses RSEM for transcript quantification and EdgeR for differential analysis. The third combines STAR, RSEM, and EdgeR for data analysis. All these workflows support multiple samples and multiple groups of samples and perform differential analysis between groups in a single workflow job submission. The calculated results are available for download and post-analysis. The supported animal species include chicken, cow, duck, goat, pig, horse, rabbit, sheep, turkey, as well as several other model organisms including yeast, C. elegans, Drosophila, and human, with genomic sequences and annotations obtained from ENSEMBL. The RNA-seq portal is freely available from http://***/RNA-seq. Conclusions: The web portal offers not only bioinformatics software, workflows, computation and reference data, but also an integrated environment for complex RNA-seq data analysis for agricultural animal species. In this project, our aim is not to develop new RNA-seq tools, but to build web workflows for using popular existing RNA-seq methods and make these tools more accessible to the communities.

关键词： RNA-seq Animal genomes Workflow Mapping Assembly transcript quantification

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Distinct expression patterns of HCN channels in HL-1 cardiomyocytes

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BMC CELL BIOLOGY 2015年第1期16卷 1-8页

作者： Guenther, Anne Baumann, Arnd Forschungszentrum Julich Inst Complex Syst Cellular Biophys ICS 4 D-52425 Julich Germany

Background: Cardiac rhythmic activity is initiated in functionally specialized areas of the heart. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are fundamental for these processes of cardiac physiology. Results: Here we investigated transcript and protein expression patterns of HCN channels in HL-1 cardiomyocytes using a combination of quantitative PCR analysis and immunocytochemistry. Gene expression profiles of hcn1, hcn2 and hcn4 were acutely affected during HL-1 cell propagation. In addition, distinct expression patterns were uncovered for HCN1, HCN2 and HCN4 proteins. Conclusions: Our results suggest that HCN channel isoforms might be involved in the concerted differentiation of HL-1 cells and may indirectly affect the occurrence of contractile HL-1 cell activity. We expect that these findings will promote studies on other molecular markers that contribute to cardiac physiology.

关键词： Pacemaker channel Cardiac cells transcript quantification Signaling Contractile activity

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Evaluation of UL99 transcript as a target for antiviral treatment efficacy

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JOURNAL OF VIROLOGICAL METHODS 2014年 207卷 104-109页

作者： Gambarino, Stefano Callea, Stefano Rizzo, Giovanna Montanari, Paola Loiacono, Elisa Bergallo, Massimiliano Univ Turin Sch Med Dept Publ Hlth & Pediat Sci Turin Italy

Human cytomegalovirus (HCMV) is a virus belonging to the Beta Herpes virus family. Its genome contains many different genes clustered in immediate early, early and late genes. This last cluster includes UL99, a late gene that encodes for a tegument protein called pp28. In immunocompetent patients, HCMV infection occurs asymptomatically, while its reactivation in immunocompromised patients can be a cause of pneumonia, retinitis and gastrointestinal diseases. To prevent or to contrast HCMV infection, several drugs (such as Ganciclovir, Acyclovir, Foscarnet) are available, and their efficiency is evaluated by HCMV DNA load monitoring, as also for antiviral resistance onset that may occur after the therapy. In this study is described the development of a Real Time PCR for the detection and quantification of UL99 transcript and the clearance of this target compared to HCMV DNA, both in vitro and in vivo on bronchoalveolar lavage samples. (C) 2014 Elsevier B.V. All rights reserved.

关键词： Human cytomegalovirus UL99 Ganciclovir Real Time PCR transcript quantification Antiviral resistance

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Digital Droplet PCR: CNV Analysis and Other Applications

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Current protocols in human genetics 2014年第1期82卷 7.24.1-7.24.13页

作者： Mazaika, Erica Homsy, Jason Department of Genetics Harvard Medical School Boston Massachusetts United States

Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. Copyright © 2014 John Wiley & Sons, Inc.

关键词： CNV copy number variant ddPCR genotyping rare variant detection TaqMan transcript quantification

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UBE3B and ZRANB1 polymorphisms and transcript abundance are associated with water holding capacity of porcine M. longissimus dorsi

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MEAT SCIENCE 2013年第2期95卷 166-172页

作者： Thi Phuong Loan Huynh Murani, Eduard Maak, Steffen Ponsuksili, Siriluck Wimmers, Klaus Leibniz Inst Farm Anim Biol FBN Inst Genome Biol D-18196 Dummerstorf Germany Leibniz Inst Farm Anim Biol FBN Inst Muscle Biol D-18196 Dummerstorf Germany Leibniz Inst Farm Anim Biol FBN Res Grp Funct Genome Anal D-18196 Dummerstorf Germany

The degradation of myofibrillar proteins during meat maturation affects the water holding capacity (WHC) of meat. Our study sought to identify polymorphisms in UBE3B and ZRANB1, genes encoding proteins involved in ubiquitination, and to evaluate the relationship between genotype, transcript abundance, and WHC of pork. A single SNP of ZRANB1, c.552A>G (***153Val), and two silent SNPs of UBE3B, c.1921A>T and c.4292C>T, were associated with muscle pH, conductivity, meat colour, or drip loss in German Landrace (GL, n = 266) and Pietrain x (Large White x German Landrace) (Pin, n = 316). Further, carriers of the minor alleles at the SNPs tended to have increased transcript abundance. Consistent with the protein degradation promoting and inhibiting effects of UBE3B and ZRANB1, respectively, and the expected impact on WHC, their expressions were positively and negatively associated with WHC. The results implicate that the SNPs in both genes are in linkage with a causal site that affects transcript abundance and WHC. (c) 2013 Elsevier Ltd. All rights reserved.

关键词： Drip loss SNP transcript quantification Real time RT-PCR Pyrosequencing Pig

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Quantifying Alternatively Spliced mRNA via Capillary Electrophoresis

Quantifying Alternatively Spliced mRNA via Capillary Electro...

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作者： Xue-Cheng Zhang Walter Gassmann

Alternative splicing (AS) significantly contributes to transcriptome and proteome complexity. transcriptome-wide studies concluded that approximately 22% of Arabidopsis and rice genes are subject to AS. Despite increasing recognition of AS in plants, little is known about the function of individual products of AS. In our studies of the Arabidopsis RPS4 resistance gene, which requires AS transcripts for function, the need to quantify AS transcripts became apparent. Because RPS4 expression levels are very low and the pattern of RPS4 splicing is complex, existing mRNA quantification methods were not adequate. We therefore developed a new method based on reverse transcription (RT) PCR amplification of all transcript variants with a common set of primers and separation of the PCR products by size via capillary electrophoresis. Products were quantified by analysis of several PCR cycles per sample using the signal quantification procedures developed for microsatellite genotyping on capillary sequencing machines. With this method, we were able to measure differential regulation of individual RPS4 alternative transcripts specifically during effector-triggered immunity. This method is especially suitable for quantification of alternative transcripts of low-expressed genes exhibiting complex splicing patterns. less

关键词： Capillary Electrophoresis (CE) MRNA Extraction PCR Reverse transcription PCR Genotyping Quantitative Reverse transcription PCR Regression Analysis RNA Extraction Data analysis Arabidopsis Alternative splicing transcript quantification RPS4 Capillary array GeneMapper Disease resistance gene

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Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy

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LABORATORY INVESTIGATION 2010年第9期90卷 1396-1402页

作者： Spitali, Pietro Heemskerk, Hans Vossen, Rolf H. A. M. Ferlini, Alessandra den Dunnen, Johan T. 't Hoen, Peter A. C. Aartsma-Rus, Annemieke Univ Ferrara Dept Expt & Diagnost Med Med Genet Sect I-44100 Ferrara Italy Leiden Univ Dept Human Genet Med Ctr NL-2300 RA Leiden Netherlands

Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains B1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. Laboratory Investigation (2010) 90, 1396-1402;doi:10.1038/labinvest.2010.98;published online 10 May 2010

关键词： antisense oligonucleotides digital array duchenne muscular dystrophy exon skipping transcript quantification

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Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references

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JOURNAL OF EXPERIMENTAL BOTANY 2009年第2期60卷 487-493页

作者： Guenin, Stephanie Mauriat, Melanie Pelloux, Jerome Van Wuytswinkel, Olivier Bellini, Catherine Gutierrez, Laurent Univ Picardie Jules Verne Ctr Mol Biol Ctr Ressources Reg Biol Mol Fac Sci 33 Rue St Leu F-80039 Amiens France Swedish Univ Agr Sci Umea Plant Sci Ctr Dept Forest Genet & Plant Physiol S-90183 Umea Sweden INRA Biol Cellulaire Lab F-80039 Versailles France Univ Picardie Jules Verne Fac Sci EA Biol Plantes & Controle Insectes Ravageurs 390 F-80039 Amiens France

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

关键词： Gene expression normalization quantitative RT-PCR reference gene transcript quantification

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Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs

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BMC Research Notes 2008年第1期1卷 1-7页

作者： Nino-Soto, Maria I. Jozani, Razi Jafari Bridle, Byram Mallard, Bonnie A. Department of Pathobiology Ontario Veterinary College University of Guelph Guelph ON N1G 2W1 Canada Faculty of Veterinary Medicine University of Tehran Tehran Iran

Background. The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells. Findings. Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation. Genes varied according to the comparison analyzed. Further testing with qPCR revealed the same trend of differential expression for 4 of the genes, although statistical significance was reached for only one. Conclusion. A new SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges. © 2008 Nino-Soto1 et al;licensee BioMed Central Ltd.

关键词： Production Trait Allele Group Swine Leukocyte Antigen transcript quantification Heterologous Hybridization

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