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The impact of SNPs on the interpretation of SAGE and MPSS experimental data.

ABSTRACT: Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS) are powerful techniques for gene expression analysis. A crucial step in analyzing SAGE and MPSS data is the assignment of experimentally obtained tags to a known transcript. However, tag to transcript assignment is not a straightforward process since alternative tags for a given transcript can also be experimentally obtained. Here, we have evaluated the impact of Single Nucleotide Polymorphisms (SNPs) on the generation of alternative SAGE and MPSS tags. This was achieved through the construction of a reference database of SNP-associated alternative tags, which has been integrated with SAGE Genie. A total of 2020 SNP-associated alternative tags were catalogued in our reference database and at least one SNP-associated alternative tag was observed for approximately 8.6% of all known human genes. A significant fraction (61.9%) of these alternative tags matched a list of experimentally obtained tags, validating their existence. In addition, the origin of four out of five SNP-associated alternative MPSS tags was experimentally confirmed through the use of the GLGI-MPSS protocol (Generation of Long cDNA fragments for Gene Identification). The availability of our SNP-associated alternative tag database will certainly improve the interpretation of SAGE and MPSS experiments.

SUBMITTER: Silva AP

PROVIDER: S-EPMC534621 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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The impact of SNPs on the interpretation of SAGE and MPSS experimental data.

Silva Ana Paula M AP De Souza Jorge E S JE Galante Pedro A F PA Riggins Gregory J GJ De Souza Sandro J SJ Camargo Anamaria A AA

Nucleic acids research 20041123 20

Serial Analysis of Gene Expression (SAGE) and Massively Parallel Signature Sequencing (MPSS) are powerful techniques for gene expression analysis. A crucial step in analyzing SAGE and MPSS data is the assignment of experimentally obtained tags to a known transcript. However, tag to transcript assignment is not a straightforward process since alternative tags for a given transcript can also be experimentally obtained. Here, we have evaluated the impact of Single Nucleotide Polymorphisms (SNPs) on ...[more]

PMID: 15562001

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Project description:BackgroundRice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods.ResultsThe MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray.ConclusionThe comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.

Project description:BACKGROUND: Metatranscriptomics is rapidly expanding our knowledge of gene expression patterns and pathway dynamics in natural microbial communities. However, to cope with the challenges of environmental sampling, various rRNA removal and cDNA synthesis methods have been applied in published microbial metatranscriptomic studies, making comparisons arduous. Whereas efficiency and biases introduced by rRNA removal methods have been relatively well explored, the impact of cDNA synthesis and library preparation on transcript abundance remains poorly characterized. The evaluation of potential biases introduced at this step is challenging for metatranscriptomic samples, where data analyses are complex, for example because of the lack of reference genomes. RESULTS: Herein, we tested four cDNA synthesis and Illumina library preparation protocols on a simplified mixture of total RNA extracted from four bacterial species. In parallel, RNA from each microbe was tested individually. cDNA synthesis was performed on rRNA depleted samples using the TruSeq Stranded Total RNA Library Preparation, the SMARTer Stranded RNA-Seq, or the Ovation RNA-Seq V2 System. A fourth experiment was made directly from total RNA using the Encore Complete Prokaryotic RNA-Seq. The obtained sequencing data were analyzed for: library complexity and reproducibility; rRNA removal efficiency and bias; the number of genes detected; coverage uniformity; and the impact of protocols on expression biases. Significant variations, especially in organism representation and gene expression patterns, were observed among the four methods. TruSeq generally performed best, but is limited by its requirement of hundreds of nanograms of total RNA. The SMARTer method appears the best solution for smaller amounts of input RNA. For very low amounts of RNA, the Ovation System provides the only option; however, the observed biases emphasized its limitations for quantitative analyses. CONCLUSIONS: cDNA and library preparation methods may affect the outcome and interpretation of metatranscriptomic data. The most appropriate method should be chosen based on the available quantity of input RNA and the quantitative or non-quantitative objectives of the study. When low amounts of RNA are available, as in most metatranscriptomic studies, the SMARTer method seems to be the best compromise to obtain reliable results. This study emphasized the difficulty in comparing metatranscriptomic studies performed using different methods.

Dataset Information

The impact of SNPs on the interpretation of SAGE and MPSS experimental data.

Publications

The impact of SNPs on the interpretation of SAGE and MPSS experimental data.

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