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rickud / gel-lanes-fit

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Don't understand how to fill 'select fragment distribution' #13

Open majdabdul opened 3 years ago

majdabdul commented 3 years ago

Dear Rick,

Thank you for creating this tool. I have run a cDNA gel and want to assess the distribution of my fragments for the purpose of (long-read) sequencing, as I don't have access to a Bioanalyzer/Tapestation.

I used the NEB QuickLoad 1 Kb extend ladder. The cDNA lane shows a smear, as expected, so I selected the continuum fit type. When I am filling out the window for gel lanes fit, I have to fill in 'select fragment length distribution'. I'm not sure what this means, how to fill it, or what the various Phage DNA options are there for. I guessed that 'Uniform' would be the option to choose for my purpose because I don't have Phage DNA and my cDNA sample is not a ladder. Then to fill the 'Distribution Range' window, I guessed based on the gel that my largest fragments (the 'upper' field) are at around 6 kb and the smallest (the 'lower' field) at around 100 bp. But what do I put for 'Every'? I'm not sure what that means or what to put there.

I would really appreciate your help.

Thank you, Majd

rickud commented 3 years ago

Hi Majd,

Yes, all your guesses are correct. The population of fragments is unknown in the case of fragmentation libraries. The assumption here was that there would be a continuous representation of many fragment lengths within the range that you expect. Every means an increase in fragment length of x bps at each step until you reach the max you selected. Then the plugin fits that putative population to the smear profile. I apologize if the documentation is not very clear yet. It's definitely a work in progress and we could use some crowdhelp updating it.

Rick

majdabdul commented 3 years ago

Hi Majd,

Yes, all your guesses are correct. The population of fragments is unknown in the case of fragmentation libraries. The assumption here was that there would be a continuous representation of many fragment lengths within the range that you expect. Every means an increase in fragment length of x bps at each step until you reach the max you selected. Then the plugin fits that putative population to the smear profile. I apologize if the documentation is not very clear yet. It's definitely a work in progress and we could use some crowdhelp updating it.

Rick

Hi Rick,

Thank you for your response! I understand better how to fill it now, but I have no idea how much of an increase in fragment lengths I have at each step. How can I guesstimate that from a smear? In case it's relevant, this library is not fragmented. Since it's for long-read sequencing (Nanopore), it's just RNA converted to cDNA and PCR'ed.

Thanks again! Majd