tophat, the parameter "Expected inner distance between mate pairs"?

Question

[Pei]

Hi:

To run tophat one has to provide a parameter: "Expected inner distance between mate pairs" with the tag -r

Suggested by 1, this parameter seemed to be relied on read length: "For example, if your fragment size is 300 bp and read length is 50 bp, the inner distance is 200"

My questions are:

1. Does this parameter mean the expected distance between two genomic loci mapped by two reads?
2. What does the "fragments size" mean and how to decide this value?

Sincerely hope that you wise guys can give me some tips.

Thanks in advance!

Best!

[Brian Bushnell]

BBMap can autodetect the insert size. After it finishes, it will print the average insert size on the screen... which, if you want, you can feed to Tophat. Or, you can just use the output of BBMap directly, since it is a splice-aware RNA-seq aligner.

Answer 1

[GouthamAtla]

Fragment size is the length of DNA sequence that is been given to sequencer. In paired-end sequencing, the DNA sequence is read from both the ends creating Read_1 and Read_2. The inner mate distance is the gap left between the two fragments after sequencing. For e.g

when 200bp fragment is read 50bp from both the sides, the gap left is 100 bp.

Read this blog: http://thegenomefactory.blogspot.in/2013/08/paired-end-read-confusion-library.html

fragment                  ========================================
fragment + adaptors    ~~~========================================~~~
insert                    ========================================
PE reads                R1--------->                    <---------R2
inner mate                           ...................