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Old 02-02-2012, 03:38 AM   #1
ulz_peter
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Location: Graz, Austria

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Default Bacterial contamination in salival sample

Hi Guys,

Now to something completely different... We would like to sequence DNA obtained from saliva. Now the thing is saliva contains a lot of bacteria (according to Wikipedia: 500 million cells / ml). When we isolate DNA their DNA should still be there, right? So, if we'd make a shotgun library we would lose quite a lot of reads to bacterial DNA being sequenced.

Is there a way to get rid of that or is that already done during DNA isolation (we're using Oragene Kit)?

Any help is highly appreciated,

Peter
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Old 02-20-2012, 11:02 AM   #2
cliffbeall
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This made me chuckle because our group is interested in the bacterial sequences from saliva. You wouldn't want to send us the bacterial sequences you're not going to use, would you?

Seriously, the only thing I can think of is to do the lysis step without a lot of agitation. A lot of bacteria are fairly tough. We use Qiagen and add bead-beating to completely recover the bacterial DNA.
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Old 02-20-2012, 11:57 AM   #3
ECO
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I have seen a couple methods out there for differential lysis of human vs bacterial cells (one was from a company called ZyGEM but i couldn't find it just now, it was a couple years ago).

My experience is that with the Oragene kit's fairly strong lysis/preservation buffer you'll get a ton of bacterial DNA as well. As cliffbeall said, you may not get perfect representation of the bacteria due to differental resistance to lysis.

Buccal swabs are cleaner, as you're scraping human tissue intentionally, but you won't get as much yield/sample.
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