RESUMO
Serial analysis of gene expression (SAGE) is a high-throughput method for global gene expression analysis that allows the quantitative and simultaneous analysis of a large number of transcripts. SAGE is a digital method and its sensitivity depends only on the number of tags sequenced. Furthermore, SAGE is a powerful tool for finding novel genes that are expressed under certain conditions or in certain tissues. SAGE has been widely used in fields as diverse as cancer research and the development and study of microorganisms. The SAGE method is a series of routine molecular biology procedure and can, at least in principle, be carried out in any laboratory. However, the number of consecutive steps is quite large and in practice, SAGE has been difficult to carry out on a routine basis.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Solanum tuberosum/genética , Transformação GenéticaRESUMO
BACKGROUND: During gene expression analysis by Serial Analysis of Gene Expression (SAGE), duplicate ditags are routinely removed from the data analysis, because they are suspected to stem from artifacts during SAGE library construction. As a consequence, naturally occurring duplicate ditags are also removed from the analysis leading to an error of measurement. RESULTS: An algorithm was developed to analyze the differential occurrence of SAGE tags in different ditag combinations. Analysis of a pancreatic acinar cell LongSAGE library showed no sign of a general amplification bias that justified the removal of all duplicate ditags. Extending the analysis to 10 additional LongSAGE libraries showed no justification for removal of all duplicate ditags either. On the contrary, while the error introduced in original SAGE by removal of naturally occurring duplicate ditags is insignificant, it leads to an error of up to 3 fold in LongSAGE. However, the algorithm developed for the analysis of duplicate ditags was able to identify individual artifact ditags that originated from rare nucleotide variations of tags and vector contamination. CONCLUSION: The removal of all duplicate ditags was unfounded for the datasets analyzed and led to large errors. This may also be the case for other LongSAGE datasets already present in databases. Analysis of the ditag population, however, can identify artifact tags that should be removed from analysis or have their tag count adjusted.
Assuntos
Etiquetas de Sequências Expressas , Regulação da Expressão Gênica/genética , Software , Algoritmos , Sequência de Bases/genética , Etiquetas de Sequências Expressas/metabolismoRESUMO
Digital transcriptomics with pyrophosphatase based ultra-high throughput DNA sequencing of di-tags provides high sensitivity and cost-effective gene expression profiling. Sample preparation and handling are greatly simplified compared to Serial Analysis of Gene Expression (SAGE). We compare DeepSAGE and LongSAGE data and demonstrate greater power of detection and multiplexing of samples derived from potato. The transcript analysis revealed a great abundance of up-regulated potato transcripts associated with stress in dormant potatoes compared to harvest. Importantly, many transcripts were detected that cannot be matched to known genes, but is likely to be part of the abiotic stress-response in potato.
