We are interested in looking at the overexpression of select transcription factors in cancer cell lines. Our RNA sequencing data from cancer tissue suggests there may be alternative splice sites in exons or UTRs of some of these genes.
Have a look at the coverage of reads in the IGV genome browser first. If the coverage has "jumps and bumps", checking it with QPCR with primers in such a location may not be reliable (not sure how the expression plasmid works - I am a bioinformatician ;)). Try to design the extra validations in the exonic regions that have a stable coverage function - you can do it separately in several regions perhaps.
You may try also to do "manual/by eye" attempt to do isoform deconvolution from the coverage from your gene of interest. Or try RSEM or Bitseq, but this may be biased by the existing annotations and statistical assumptions.
As for the UTRs - it may to be tricky, as the UTR has often a gradually decreasing pattern of expression (as coverage function in RNAseq). Again - if the coverage of RNAseq is stable within the UTR or part of it, chances are that the extra vaidation method will be reliable.
Actually - you may cut out with samtools your gene to make a small BAM and email me together with your reference sequence - I can try to foretell what is the case ;) but it is never guaranteed that this coverage will be informative enough
Thanks Michael. We will amplify regions with good coverage on IGV by qPCR and use those PCR products for cloning. We haven't done enough cloning to know if adding the UTRs might make overexpression more difficult.
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