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  • Basic question on read count

    In the ENCODE RNA-seq guidelines it states:
    "30M pair-end reads of length > 30NT, of which 20-25M are mappable to the genome or known transcriptome"
    and
    "For experiments from a typical mammalian tissue or in which sensitivity of detection is important, a minimum depth of 100-200 M 2 x 76 bp or longer reads is currently recommended. "

    My question: The 30 million they refer to is counting both reads in a pair individually or do they count one pair as one read?

    So, say I decide on a read length of 100. The amount of data I will require(or ask from a provider) is:
    (A) 100(read length) X 30,000,000 = 3Gb
    Or if I count each pair as one i.e 30 million pairs
    (B) 100(read length) X 2 (for each pair) X 30,000,000 = 6 Gb

    Should it be (A) or (B)?

    PS: I know the question is basic and I tried to do a search but couldn't find a definite answer.

  • #2
    Well, it's just a guideline. More is better. And while the guideline is ambiguous, it does not really matter whether you sequence precisely 100M reads rather than 75M; you'll just get slightly more complete data in the low-abundance genes. At 200M you'll get some genes that you would miss at 100M, but at 400M you'd get some that you would miss at 200M.

    For quantification of gene expression, it's the number of fragments that is statistically important (meaning number of reads, if single-ended, or read pairs, if paired). The length or whether they are paired is not important as long as they are long enough to map uniquely. For relative isoform abundance, though, the length and paired-ness is important. In other words, if you don't care about alternative splicing, and you can get 200M single-ended 100bp reads for the same price as 100M pairs of 100bp reads, the second is better.

    That said - the sentence literally means 100M-200M reads, meaning 50M-100M pairs, regardless of what the author may have intended.

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