Browsing by Author "Pan, Cheng-Tsung"

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A simple method using Pyrosequencing (TM) to identify de novo SNPs in pooled DNA samples
(2011-03-01)
Lin, Yeong-Shin
;
Liu, Fu-Guo Robert
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Wang, Tzi-Yuan
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Pan, Cheng-Tsung
;
Chang, Wei-Ting
A practical way to reduce the cost of surveying single-nucleotide polymorphism (SNP) in a large number of individuals is to measure the allele frequencies in pooled DNA samples. Pyrosequencing (TM) has been frequently used for this application because signals generated by this approach are proportional to the amount of DNA templates. The Pyrosequencing (TM) pyrogram is determined by the dispensing order of dNTPs, which is usually designed based on the known SNPs to avoid asynchronistic extensions of heterozygous sequences. Therefore, utilizing the pyrogram signals to identify de novo SNPs in DNA pools has never been undertook. Here, in this study we developed an algorithm to address this issue. With the sequence and pyrogram of the wild-type allele known in advance, we could use the pyrogram obtained from the pooled DNA sample to predict the sequence of the unknown mutant allele (de novo SNP) and estimate its allele frequency. Both computational simulation and experimental Pyrosequencing(TM) test results suggested that our method performs well. The web interface of our method is available at http://life.nctu.edu.tw/similar to yslin/PSM/.
Evaluation and Application of the Strand-Specific Protocol for Next-Generation Sequencing
(2015-01-01)
Tsai, Kuo-Wang
;
Chang, Bill
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Pan, Cheng-Tsung
;
Lin, Wei-Chen
;
Chen, Ting-Wen
Next-generation sequencing (NGS) has become a powerful sequencing tool, applied in a wide range of biological studies. However, the traditional sample preparation protocol for NGS is non-strand-specific (NSS), leading to biased estimates of expression for transcripts overlapped at the antisense strand. Strand-specific (SS) protocols have recently been developed. In this study, we prepared the same RNA sample by using the SS and NSS protocols, followed by sequencing with Illumina HiSeq platform. Using real-time quantitative PCR as a standard, we first proved that the SS protocol more precisely estimates gene expressions compared with the NSS protocol, particularly for those overlapped at the antisense strand. In addition, we also showed that the sequence reads from the SS protocol are comparable with those from conventional NSS protocols in many aspects. Finally, we also mapped a fraction of sequence reads back to the antisense strand of the known genes, originally without annotated genes located. Using sequence assembly and PCR validation, we succeeded in identifying and characterizing the novel antisense genes. Our results show that the SS protocol performs more accurately than the traditional NSS protocol and can be applied in future studies.
Global Investigation of Immune Repertoire Suggests Kawasaki Disease Has Infectious Cause
(2019-10-01)
Kuo, Ho-Chang
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Pan, Cheng-Tsung
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Huang, Ying-Hsien
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Huang, Fu-Chen
;
Lin, Yeong-Shin
Background: Kawasaki disease (KD) severely threatens young children's health worldwide. The pathogenic mechanism of KD has not yet been solved, so there is still debate over whether KD is an infectious disease or an autoimmune disease. Methods and Results: To solve this problem, an immune repertoire analysis of KD was conducted. We collected blood cell RNA samples and prepared them into amplicons with iRepertoire kits. The amplicons were sequenced and analyzed with the iRepertoire pipeline. We first identified KD-specific VJ and VDJ forms that had the potential to serve as biomarkers of KD. In addition, the KD-specific VDJ forms were contributed mostly by immunoglobulin G. The D50 value analysis showed that B-cell diversity in KD is decreased, suggesting unique immunoglobulins are produced in KD. Moreover, V, D and J segment usage in IgA, IgG and IgM was consistent with previous KD studies. Further comparison showed no difference in CDR3 peptide length between KD and fever controls (subjects with fever but not diagnosed as KD), indicting KD had B-cell selection phenomenon that has a non-autoimmune pattern. The comparison of amino acid usage of the CDR3 region demonstrated a preference for hydrophilic amino acids in KD. Conclusions: The results of D50 value, VDJ usage and CDR3 peptide length analyses suggested the characteristics of infectious disease for KD.
miRSeq: A User-Friendly Standalone Toolkit for Sequencing Quality Evaluation and miRNA Profiling
(2014)
Pan, Cheng-Tsung
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Tsai, Kuo-Wang
;
Hung, Tzu-Min
;
Lin, Wei-Chen
;
Pan, Chao-Yu
MicroRNAs (miRNAs) present diverse regulatory functions in a wide range of biological activities. Studies on miRNA functions generally depend on determining miRNA expression profiles between libraries by using a next-generation sequencing (NGS) platform. Currently, several online web services are developed to provide small RNANGS data analysis. However, the submission of large amounts of NGS data, conversion of data format, and limited availability of species bring problems. In this study, we developed miRSeq to provide alternatives. To test the performance, we had small RNA NGS data from four species, including human, rat, fly, and nematode, analyzed with miRSeq. The alignments results indicate that miRSeq can precisely evaluate the sequencing quality of samples regarding percentage of self-ligation read, read length distribution, and read category. miRSeq is a user-friendly standalone toolkit featuring a graphical user interface (GUI). After a simple installation, users can easily operate miRSeq on a PC or laptop by using a mouse. Within minutes, miRSeq yields useful miRNA data, including miRNA expression profiles, 3\' end modification patterns, and isomiR forms. Moreover, miRSeq supports the analysis of up to 105 animal species, providing higher flexibility.
Multiomics analyses identified epigenetic modulation of the S100A gene family in Kawasaki disease and their significant involvement in neutrophil transendothelial migration
(2018-11-01)
Huang, Lien-Hung
;
Kuo, Ho-Chang
;
Pan, Cheng-Tsung
;
Lin, Yeong-Shin
;
Huang, Ying-Hsien
BackgroundKawasaki disease (KD) is a prevalent pediatric disease worldwide and can cause coronary artery aneurysm as a severe complication. Typically, DNA methylation is thought to repress the expression of nearby genes. However, the cases in which DNA methylation promotes gene expression have been reported. In addition, globally, to what extent DNA methylation affects gene expression and how it contributes to the pathogenesis of KD are not yet well understood.MethodsTo address these important biological questions, we enrolled subjects, collected DNA and RNA samples from the subjects' total white blood cells, and performed DNA methylation (M450K) and gene expression (HTA 2.0) microarray assays.ResultsBy analyzing the variation ratios of CpG beta values (methylation percentage) and gene expression intensities, we first concluded that the CpG markers close (-1500bp to +500bp) to the transcription start sites had higher variation ratios, reflecting significant regulation capacities. Next, we observed that, globally speaking, gene expression was modestly negatively correlated (correlation rho approximate to-0.2) with the DNA methylation status of both upstream and downstream CpG markers in the promoter region. Third, we found that specific CpG markers were hypo-methylated in disease samples compared with healthy samples and hyper-methylated in convalescent samples compared with disease samples, promoting and repressing S100A genes' expressions, respectively. Finally, using an in vitro cell model, we demonstrated that S100A family proteins enhanced leukocyte transendothelial migration in KD.ConclusionsThis is the first study to integrate genome-wide DNA methylation with gene expression assays in KD and showed that the S100A family plays important roles in the pathogenesis of KD.