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  • Probability of sequencing low abundant transcripts from non-normalized library

    Dear users,

    I am new to this community and this is my first thread, I am also new to next generation sequencing analysis but I have interesting issue and I want to get some feedback from you. Thanks in advance for any replies!

    Here is a scenario:
    > non-normalized eukaryotic transcriptome library
    > 3' Tag Sequencing; Reads of 100nts length; Illumina HiSeq 2000 Single Reads Machine

    Two sequencing Attempts:
    1) 50 Million Reads at one run with starting RNA consentration X
    2) 3 Runs with respectively 10MilReads, 20MilReads, 20 MilReads with starting RNA concentration X

    The RNA concentration is the same in the sample before any of the four runs!

    Question: Do I get the same set of transcriptome representation between attempt 1 and attempt 2?

    Note: Assuming that the probability of getting a very low abundant transcript is constant between runs, i would expect that the chance of observing these transcript in a smaller sampling size is less than in a higher sampling size

    Predicted observation: In the second attempt after adding results I would expect that the transcriptome is overwhelmed only by transcripts of higher concentration in the sample, while I would expect to get more less abundant transcrips in the one time sequencing run with 50MilReads (attempt 1)

    Thank you for your discussions!

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