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Old 05-09-2013, 03:30 PM   #1
Seqanalysis
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Default Completing genome of thermophilic bacteria

Hi,
I sequenced the genome of a thermophilic bacteria using Illumina. There was a gap of 37kb that was missed during sequencing but I was able to fill that using sanger sequecing. This region seemed to be highly conserved but has 33genes that are designated as hypothetical proteins.
My problem is I am not able to sequence a gene which is about 3.8kb completely. With internal pair of primers i was able to get 1.9kb sequence but am not able to walk in using primers from neighboring genes which is strange as I know the genes are together.
Can anyone suggest me what could be the possibilities?

This region is probably not stable in this organism it seems to be susceptible to degradation during extraction.
What could be the reasons?
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Old 05-09-2013, 08:07 PM   #2
ECO
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Super high GC? Repetitive elements? Gene family? N4-mC content interfering with PCR?

Sounds like a job for a couple PacBio SMRTcells*


*disclaimer: I used to work there. But this is textbook RS/RSII.
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Old 05-10-2013, 06:55 AM   #3
Seqanalysis
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GC is 46% for my organism. Surprisingly I get the pcr products in other species that I use as control. This region is highly conserved in this genus. Repetitive seq yes they have a lot but then again it's the same in other species.

I really appreciate your suggestions. I would be glad to know more
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