Supplementary MaterialsAdditional data file 1 Shown are examples of RACE PCR

Supplementary MaterialsAdditional data file 1 Shown are examples of RACE PCR products on an agarose gel. Clearly, lowering the threshold will increase the sensitivity of our analysis, while decreasing the specificity. gb-2008-9-1-r3-S2.pdf (3.0K) GUID:?C85E41E8-4856-47EB-AD2E-45A000BA42AA Additional data file 3 Further explanation of consensus splice site analyses : In order to decide the window size for the consensus splice site analysis, we considered a simplified model in which a nucleotide sequence of length em N /em is generated by randomly selecting A, C, G, T with equal probability of 1/4, and then computed the probability ( em prob_pattern /em ) of that sequence containing at least one pattern of a consensus splice site (for example, having either ‘GT’ or ‘AG’ in the sequence). This is as follows: em prob_pattern /em ( em N /em ) = em count_pattern /em ( em N /em )/(4^ em N /em ), where em count_pattern /em (1) = 0, em count_pattern /em (2) = 1, em count_pattern /em ( em N /em ) = 4^( em N /em – 2) – em count_pattern /em ( em N /em – 2) + 4 em count_pattern /em ( em N /em – 1), for em N /em 2. Although this formula does not take into account many sophisticated factors in reality, it can provide us a good guideline on selecting the window size for our analysis. This file shows the squared values of such probabilities (which NVP-LDE225 cost can be considered as a lower bound of the probability for a random sequence to have a complete consensus pattern) for em N /em ranging from 2 to 13. In the analysis of this paper, we selected the window size to be 8 to make sure at least twofold enrichment in the amount of sequences that people identified weighed against that in the simplified model, provided the same amount of sequences. gb-2008-9-1-r3-S3.pdf (4.1K) GUID:?0DF36EF5-70EC-4648-BB94-5C7F430E0983 Extra data file 4 This file could NVP-LDE225 cost be uploaded towards the University of California at Santa Cruz Genome Brower to see all RACE products. gb-2008-9-1-r3-S4.bed (50K) GUID:?9D1BF93D-F76C-4FC2-9812-4929B7E25788 Abstract Background Recent studies from the mammalian transcriptome NVP-LDE225 cost have revealed a lot of additional transcribed regions and extraordinary complexity in transcript diversity. Nevertheless, there continues to be very much doubt concerning what part of the genome can be transcribed exactly, the exact constructions of these novel transcripts, and the levels of the transcripts produced. Results We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins. Conclusion We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional. Background Recent studies [1-5] have revealed that the composition and structure of the mammalian transcriptome is much more complex than was previously thought. Large-scale RT-PCR analysis to determine the structure of transcripts produced from exons of known human genes has shown that multiple transcripts are produced from most gene loci (an average of more than five was reported by Harrow and coworkers [6]). In many cases Mertk the 5′ ends of these alternate transcripts are located more than 100 kilobases upstream from the previously known start site [1]. Likewise, systematic analysis of cloned mouse and human cDNAs revealed that many even more transcripts than previously valued are transcribed from each known gene locus [7-9]. One way to obtain complexity can be substitute 5′ ends; latest studies indicate that we now have at least 36% even more promoters than once was recognized [10-14]. As well as the variety of transcripts from known loci, it would appear that much more from the human being genome can be transcribed than once was valued. Probing of tiling arrays with cDNA probes offers indicated that we now have at least doubly many transcribed areas.

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