Does RNA-Seq sequence randomly?

[bob-loblaw]

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?

[westerman]

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.

[maubp]

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.

[bob-loblaw]

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.

Okay thanks, it was an illumina hi-seq machine