Mean insert size and Mean inner distance between mate pairs

Question

[manekineko]

Hi,

I'm confused are these two the same or different values?

I have tun Picard for calculate Mean Insert size of subset of my RNA-seq FASTQ mapped to cDNA (100bp reads) and it gives me:

MEDIAN_INSERT_SIZE         176
MEDIAN_ABSOLUTE_DEVIATION  39
MIN_INSERT_SIZE            12
MAX_INSERT_SIZE            6467
MEAN_INSERT_SIZE           193.140364

So what value should I feed to Tophat - Mean inner distance between mate pairs? 193 or?

And then somewhere I saw which confused me more ---> Mean inner distance between mate pairs = mean insert-size-2*read_length

Answer 1

[Brian Bushnell]

Mean inner distance between mate pairs = mean insert-size-2*read_length

That is exactly correct.

So for a 2x100bp library with 193 mean insert, your inner distance is 193-2x100 = -7 (meaning that they overlap by 7bp).

[manekineko]

So I should use a negative -7 in Tophat (the default says it is 50)? is negative value ok?

[Brian Bushnell]

It should be fine. A negative inner distance is very common. If it doesn't like -7 just use 0 which is close enough; it doesn't need to be exact.