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Old 02-24-2016, 04:47 PM   #1
flef
Junior Member
 
Location: Canada

Join Date: Feb 2016
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Default Finding T-DNA insertion events in complex genome

Hi, I need to design a sequencing experiment to locate about a dozen insertion events of a ~10kbp transgene of known sequence in a very complex plant genome (~10Gpb, polyploid). I have access to Illumina HiSeq, MiSeq and PacBio RS machines. The T-DNA may have inserted in tandem repeats and may have internal rearrangements.

The original proposal was targeted capture followed by PacBio sequencing. Do you think this is feasible?

If we decide to go for whole genome sequencing, do you think long insert mate pairs (3 or 5 or 10kbp) would help?

Thanks!

EDIT: I forgot to mention the genome is poorly characterized, and that we would like to obtain about the sequences flanking the insertions, say 1kbp.

Last edited by flef; 02-24-2016 at 04:57 PM.
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Old 02-25-2016, 03:33 AM   #2
nucacidhunter
Jafar Jabbari
 
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Long fragment capture followed by PacBio is a good option and cost effective. You are less likely to obtain 1 kb of flanking regions with mate pairs.
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insertion, mates, pacbio, t-dna, transgene

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