Hi 1_GOld,

This can be very confusing when you first start, but the fragment length is not the same as the read length.

When sequencing is conducted, the nucleic acids are fragmented - this can be done physically, using something like sonication, enzymically, using a nuclease of some sort (e.g. RNAse I in Ribo-seq) or the library can be generated using a randomly inserting transposase, as in tagmentation. Either way, you are left with a collection of randomly generated "fragments" of the original molecule. Sequencing adaptors are then added to the ends of these fragments, and a fixed number of nucleotides is sequenced from one (single-end) or both (paired-end) ends of the fragment. Usually only a portion of the fragment is sequenced, and we rarely have the full sequence of a fragment.

So in RNA-seq a full length cDNA might be broken up so:

|>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>|
                                 | framgementation
                                 V
|>>>>>>>|  |>>>>>>>>>>>>>>>>>>>>|  |>>>>>>>>>>>>>>>>>>>>>>>>| |>>>>>|
                                 | adaptor ligation
                                 V
--|>>>>>>>|--    --|>>>>>>>>>>>>>>>>>>>>|--    --|>>>>>>>>>>>>>>>>>>>>>>>>|--   --|>>>>>|--
                                 |   Single end unstranded sequencing
                                 V
|->>>>>| 100nt               100nt |<<<<<-|    |->>>>>| 100nt                100nt |<<<<<-|
--|>150bp>|--    --|>>>>>>>300bp>>>>>>>>|--    --|>>>>>>>>>350bp>>>>>>>>>>|--   --|120bp|--

In the above, the fragments are 150, 300, 350 and 120bp in length (mean: 230, sd: 112), but the reads are all 100nt long.

You can have a guess at the fragment size distribution using the QC (e.g. a tape station or bioanalzyer run) from the library prep if you have it - if you had your library sequenced commercially, they might have provided this. In the days of full alignment based workflows, there were ways of estimating this from the alignments, but for alignment-free or pseudo-alignment methods, you will need to guess if you don't have the QC.