Hi everyone,
I'm currently trying to optimise a ddradtag protocol and after we received our first set of sequences back, it appears we've had a few issues. (We are using Stacks to analyse our data)
My first question is, when using EcoRI as one of the digest enzymes, does anyone know if there should be a difference in fragment numbers depending on which size you select? We are going mostly off the Peterson paper and according to them, the fragment distribution appears to be fairly uniform.
We have size selected for ~400bp (+/- 50bp) which I know is quite a large window, but we are getting fairly excessive numbers of stacks (upwards of 75000) with quite a low depth.
I'm thinking its fairly likely that this is due to our wide size selection range, but as other people seem to be selecting smaller fragments, I was wondering if this is likely to have any effect?
Any advice would be appreciated.
Cheers
I'm currently trying to optimise a ddradtag protocol and after we received our first set of sequences back, it appears we've had a few issues. (We are using Stacks to analyse our data)
My first question is, when using EcoRI as one of the digest enzymes, does anyone know if there should be a difference in fragment numbers depending on which size you select? We are going mostly off the Peterson paper and according to them, the fragment distribution appears to be fairly uniform.
We have size selected for ~400bp (+/- 50bp) which I know is quite a large window, but we are getting fairly excessive numbers of stacks (upwards of 75000) with quite a low depth.
I'm thinking its fairly likely that this is due to our wide size selection range, but as other people seem to be selecting smaller fragments, I was wondering if this is likely to have any effect?
Any advice would be appreciated.
Cheers
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