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Research on the Transcriptome of Human Though RNA-Seq

Author: ChenChao
Tutor: LiuXinXing
School: Central South University
Course: Biological Engineering
Keywords: RNA-Seq transcriptome SOAP alternative splicing SNP
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 1289
Quote: 5
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Abstract


The next generation sequencing (NGS) technology such as the Illumina,454 and SOLiD platforms has emerged as a cutting edge approach over the past few years, it has dramatically improved the speed of gene discovery, analysis of methylation patterns, and identification of SNPs (single nucleotide polymorphisms). Here, we described a pipeline of studying gene expression levels, refinement of gene structure, and detection of novel transcripts, alternative splicing, SNP detection with human transcriptome based on RNA-Seq technology.We used the RNA-Seq data of human for the analysis. First, we filtered the low-quality reads and the reads containing adaptors and obtained the clean reads. Then we used the clean reads to do the further analysis. We aligned the reads to the reference gene and genome using SOAP software, The results of alignment were used for the subsequent analysis such as calculation of gene expression level, gene structure refinement and so on. The gene expression level was calculated by the RPKM (reads per kb per million reads) method, this method is able to eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression. Aimed to know the main biological process that the different expressed gene participated, we performed the GO (gene ontology) and pathway enrichment analysis based on the hypergeometric distribution. By aligning the clean reads to the reference genome using TopHat software, we acquired the junction alignment reads and then used this junction information to detect alternative splicing events. Finally, we used SOAPsnp to detect the SNPs based on the result of SOAP alignment results. Totally, we obtained 3.6Gb and 3.7Gb data for sample YZ1 and YZ2 respectively. After filtering, the base quality is quite high (Q20 are 91% and 90%, respectively). The mapped ratio for gene are both reached about 60%, and the mapped ratio for genome are both reached about 90%, indicating a high quality sequence. We detected 15770 and 15828 genes expressed in sample YZ1 and YZ2, respectively. There are 288 genes show significant differential expression between the two samples. By comparing the alignment results with the gene annotation file, we refined the gene structure and detected some novel transcripts in the samples. Totally, about 13000 genes were indentified to associated with alternative splicing, 53260 and 56117 SNP sites were detected in sample YZ1 and YZ2, respectively.In conclusion, the method we presented in this paper can help us to detect novel transcripts in the sample, find the different expressed genes (DGEs) between samples and detect variant genes generated by alternative splicing. We also enable to detect SNPs in samples that might associate with some disease. We anticipate that with the development of sequencing technology, individual genome age will come soon.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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