Question: Insert Size Mean And Sd For Sanger Reads
0
gravatar for jli99
7.0 years ago by
jli99150
jli99150 wrote:

I have some sequencing reads generated by Sanger sequencing. They are for a few BACs. How can I get the mean and SD for the insert size?

sanger • 1.4k views
ADD COMMENTlink written 7.0 years ago by jli99150
1

Do you have a reference genome against which these reads are/can be mapped?

ADD REPLYlink written 7.0 years ago by Gaffa490

No, I have no reference sequence.

ADD REPLYlink written 7.0 years ago by jli99150

Are you talking about shotgun sequencing of few BACs, or BAC-ends sequencing?

ADD REPLYlink written 7.0 years ago by Darked894.2k
1
gravatar for Sean Davis
7.0 years ago by
Sean Davis24k
National Institutes of Health, Bethesda, MD
Sean Davis24k wrote:

Assuming that you mean that you are sequencing BAC end pairs, just use blat (or another alignment algorithm) to align the reads to the genome. From there, you can simply find the distances between pairs and load those into excel, for example, to get the average and standard deviation of the distances. If this doesn't make sense for your experiment, then you may need to clarify what you are trying to do with more detail.

ADD COMMENTlink written 7.0 years ago by Sean Davis24k

I don't have the genome sequence. Some people told me to ask people who prepared the insert library and just use an approximate number for the assembling. Is this the proper way to get and use insert sizes?

ADD REPLYlink written 7.0 years ago by jli99150
1
gravatar for Darked89
7.0 years ago by
Darked894.2k
Barcelona, Spain
Darked894.2k wrote:

Assuming shotgun sequencing using Sanger:

  • during DNA sharing on some machines there are settings which will give you rough numbers, I think

  • before subcloning step everybody does some size checking, so you will have either agarose gel picture or fragment size plot.

  • if all else fails, try to find a related genome, blast the fragments and estimate the sizes that way.

ADD COMMENTlink written 7.0 years ago by Darked894.2k
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