What Are The Proper Definitions For The Terms Reads And Hits In The Context Of An Rna-Seq Analysis?

Question

[alittleboy]

Would anyone provide a brief description of "Reads" and "Hits" from a typical RNA-Seq output? I think in constructing the count table to be used for testing differentially expressed genes, the "Reads" should be use instead of Hits. What is the difference between these two? I once heard that "Hits" only tell you how many "Reads" are associated with each transcript, but "Hits" is NOT a metric for expression... Thanks!

Answer 1

[Mikael Huss]

"Reads" refers to sequences from the sequencing instrument, "hits" are mappings of a read to some genomic region (transcript, gene, exon ...). That is, a read "hits" some defined region (or several, in the case of "multi-hits" or "multi-mappers".

So you are correct that the number of hits tells you how many reads are associated with a transcript (or gene / exon / ...). It is not a direct measure of expression because the number of hits you get will depend not only on the expression level but also on (notably) the length of the transcript and how much you have sequenced. However, you can usually use the number of hits - the "count" as input to differential expression programs, because they deal with these issues internally.