Discarding duplicate ditags in LongSAGE analysis may introduce significant error

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

A human glomerular SAGE transcriptome database

Background: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. Description: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. Conclusions: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression

Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

<p>Abstract</p> <p>Background</p> <p>Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (<it>Brassica napus </it>ssp. <it>napus)</it>. The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of <it>B. napus </it>by applying a tag-to-gene matching strategy based on <it>Brassica </it>ESTs and the annotated proteome of the closely related model crucifer <it>A. thaliana</it>.</p> <p>Results</p> <p>Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by <it>Brassica </it>ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during <it>B. napu</it>s seed development.</p> <p>Conclusion</p> <p>This study underlines the potential of transcript tagging approaches for gene expression profiling in <it>Brassica </it>crop species via EST matching to annotated <it>A. thaliana </it>genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species.</p

Identification of novel androgen-responsive genes by sequencing of LongSAGE libraries

<p>Abstract</p> <p>Background</p> <p>The development and maintenance of the prostate is dependent on androgens and the androgen receptor. The androgen pathway continues to be important in prostate cancer. Here, we evaluated the transcriptome of prostate cancer cells in response to androgen using long serial analysis of gene expression (LongSAGE) libraries.</p> <p>Results</p> <p>There were 131 tags (87 genes) that displayed statistically significant (p ≤ 0.001) differences in expression in response to androgen. Many of the genes identified by LongSAGE (35/87) have not been previously reported to change expression in the direction or sense observed. In regulatory regions of the promoter and/or enhancer regions of some of these genes there are confirmed or potential androgen response elements (AREs). The expression trends of 24 novel genes were validated using quantitative real time-polymerase chain reaction (qRT-PCR). These genes were: <it>ARL6IP5, BLVRB, C19orf48, C1orf122, C6orf66, CAMK2N1, CCNI, DERA, ERRFI1, GLUL, GOLPH3, HM13, HSP90B1, MANEA, NANS, NIPSNAP3A, SLC41A1, SOD1, SVIP, TAOK3, TCP1, TMEM66, USP33</it>, and <it>VTA1</it>. The physiological relevance of these expression trends was evaluated <it>in vivo </it>using the LNCaP Hollow Fibre model. Novel androgen-responsive genes identified here participate in protein synthesis and trafficking, response to oxidative stress, transcription, proliferation, apoptosis, and differentiation.</p> <p>Conclusion</p> <p>These processes may represent the molecular mechanisms of androgen-dependency of the prostate. Genes that participate in these pathways may be targets for therapies or biomarkers of prostate cancer.</p

Gene expression profiling of mesenchymal stem cells aged in vitro

Mesenchymal Stem Cells (MSC’s) have shown promise as a cell-based therapy for myocardial repair. However, MSC’s have a finite replicative lifespan and lose proliferative and differentiation capacity during expansion in vitro. Therefore, understanding the molecular mechanisms that regulate ageing and senescence of MSC’s should enhance our ability to use these cells in cell-based approaches and give insight into mechanisms of tissue ageing. We established MSC cultures from the sternal bone marrow of eight donors undergoing coronary artery bypass surgery. After thirty population doublings (nine passages) MSC’s displayed morphological abnormalities, expression of senescence associated β-galactosidase, telomere erosion and decreased adipogenic and osteogenic differentiation capacity. Using serial analysis of gene expression (SAGE) we identified 243 known genes differentially expressed between MSC’s at passages two and nine. Analysis of known direct interactions between genes revealed a regulatory signaling network centered on down-regulation of the transcription factor, activator protein 1 (AP-1). Transcriptional changes in MSC’s at passage nine included genes associated with inflammation, regulation of cell cycle, metabolism and extracellular matrix re-modelling. The validation studies corroborated the SAGE results and eighteen genes were identified as differentially expressed in late passage MSC’s from multiple donors. Furthermore, caveolin 1, cyclin D1, tissue plasminogen activator and olfactomedin-like 3 were able to discriminate MSC’s of different culture age. In addition, we show evidence that the p38 MAPK signalling pathway contributes to the decline in proliferation and differentiation of MSC’s during expansion and is critical for the maintenance of genomic stability. The results provide further evidence that MSC’s senesce prematurely in response to undefined culture stresses. Our studies have provided novel markers that identify MSC ageing in vitro and suggest that identifying factors that activate p38 MAPK signalling should enhance our ability to use MSC’s in cell-based therapies