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  • Need Advise on the mRNA sequencing

    Hi Everyone,

    Currently a beginner in my PHD and wondering can anyone help me on this? i'm totally confuse.

    I understand that next generation sequencing sequence whole transcript length of mRNA and can be use for comparative analysis between different samples. As a novice to this new technique I do not actually quite understand the principle of this sequencing read out. I have studied the powerpoint slides provided by the illumina and we actually excised a gel slice of a product of interest e.g. 200bp which subsequently is used for PCR. The question here is what happen to the rest of the fragmented clusters (e.g. >200bp)? Don't we need to sequence those clusters as well to get the whole mRNA transcript readout? It seems to me that the 200bp product clusters provide sufficient sequence to generate the whole length transcript? Totally

    Thank you so much for the help

  • #2
    If you optimized your RNA fragmentation to get a peak centered on 200bp, you should be covering almost all of the transcriptome to some extent by cutting out a band at 200+/-25bp. Realize that what seems like a relatively small amount of input mRNA (~100ng) is actually billions of molecules and by fragmenting into small pieces and sequencing those, chances are that every transcript had equal chances of being covered. I made a spiked library recently with a 1 picogram of a 1kb RNA spike and got 3500 reads mapping to it!

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