Insert Size Mean And Sd For Sanger Reads

Question

[jli99]

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?

[Gaffa]

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

[jli99]

No, I have no reference sequence.

[Darked89]

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

Answer 1

[Sean Davis]

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.

[jli99]

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?

Answer 2

[Darked89]

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.