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i.e. if I were to select the first 1000 reads in a fastq file of RNA-Seq data, could they be considered 1000 random reads?
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Depends on which sequencing technology you are using and where your file came from. In general ... if the reads are directly off of a sequencer then the reads will be random in terms of the molecules sequences but will be ordered in terms of where on the plate/slide/etc. the sequencing occurred. Thus if there are problems with the sequencer (bubbles, poor optics, etc.) you can see non-random effects. -
So no, the first (and last reads) in the file will usually be from the edge of the plate/slide/tile and therefore be worse quality than average.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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