In Dnmt1 MEFs, the HoxC cluster was covered by a large bloc

Figure 1 shows that saturation was reached at around two million tags for most of the data sets which the distinct tags were mapped to, although the slope for the total number of distinct tags decreases without saturating. It is worth noting that all the reference transcriptome INNO-406 Bafetinib sets performed similarly, achieving a max imum near 20,000 mapped tags. However, when looking at how many distinct tags map to any of those transcrip tomes, about 5,000 tags appear not to be shared among all of them. A critical point in this kind of experiment has to do with the number of times a tag has to be seen so that it can be considered reliable. Discarding too many tags in an attempt to increase reliability will result in a loss of infor mation whereas keeping all of them may generate back ground noise.<br><br> To estimate the specificity Lapatinib Tykerb of our tags and to establish an optimal cutoff for the minimum number of counts a tag should have in order not to be considered artefactual, we performed a series of simulations mapping iteratively randomized sets of our data. The results are summarized in Additional file 3 for the different cutoffs tested. For cutoffs higher than five there is no substantial gain in terms of specificity. Thus, we defined reliable tags as those sequenced five times or more and discarded the rest. Thereafter, for the subsequent computational and experimental analyses, only those tags occurring at least five times were considered. From the initial set of 98,156 distinct tags, 40,670 passed that cutoff.<br><br> The low technical variability of DGE and its high reproducibility, together with the digital quantification of transcripts, enables direct comparison of samples purchase Lonafarnib across different experiments, even from different laboratories. That property allowed us to contrast our results with those from Galloni, who used DGE to identify neoblast genes by comparing irradiated ver sus control animals over the same strain of clonal S. mediterranea. A Venn diagram showing the similarity of the strategies can be seen in Additional file 4. From the total distinct tags, 31. 38% were sequenced 10 times or more in our study, compared with just 11,28% in the irradiation strategy, indicating a greater representation of each tag. This suggests, as expected, that the cell sorting approach has higher specificity. In addition, the strand specific nature of DGE allows the discrimination of sense and antisense transcripts.<br><br> Almost 30% of the transcripts suc cessfully identified also presented antisense transcription, even though at lower levels than canonical transcription. This confirms the findings of the aforementioned study in planarians and others, and shows that a large pro portion of the genome is transcribed from both strands of the DNA. Although the purpose of these transcripts is still open to debate, evidences point to a post transcriptional gene regulatory function. Tag mapping to reference sequence data sets An essential step in DGE is the recovery of the transcript represented by each tag. The nature of the DGE method ology, which generates reads of only 21 nucleotides, implies mapping short reads against a reference genome or a collection of ESTs to retrieve full length sequences for the original transcripts.