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-   -   16S-V4 library peak at smaller bp in bioanalyzer trace (http://seqanswers.com/forums/showthread.php?t=70999)

yuehuang 08-22-2016 11:59 AM

16S-V4 library peak at smaller bp in bioanalyzer trace
 
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Dear All,

I used 16S-V4 kit(BIOO Scientific) to make libraries of DNA extracted from fresh plasma. But the dominant peak is at 350bp, instead of 450bp as suggested by the kit protocol. It's my first time to use this kit. Would anyone kindly help me what this peak could be and what I should do to make it correct?

BTW: the input DNAs were all below 5ng, so I strictly follow the protocol the manufacturer provided and set the PCR II cycles at 22.

Thanks much!

Yue

Bioo Scientific 08-23-2016 01:25 PM

Hi Yue,

We would be happy to help you with this. Can you contact Bioo Scientific’s technical service at BiooNGS@biooscientific.com or 512-707-8993?

Regards,
Bioo Scientific

yuehuang 08-23-2016 01:37 PM

Thanks much!

I'm discussing this with Brad, your technical support. He's helpful, I believe we will figure this out.


Quote:

Originally Posted by Bioo Scientific (Post 198115)
Hi Yue,

We would be happy to help you with this. Can you contact Bioo Scientificís technical service at BiooNGS@biooscientific.com or 512-707-8993?

Regards,
Bioo Scientific


adam.geber 08-24-2016 07:34 AM

yuehuang, would you mind sharing your findings here when appropriate? I use a different method to make 16S libraries but have experienced similar sizing discrepancies in the past so I'm interested to know how you and Bioo interpret these results.

yuehuang 08-24-2016 02:41 PM

Quote:

Originally Posted by adam.geber (Post 198169)
yuehuang, would you mind sharing your findings here when appropriate? I use a different method to make 16S libraries but have experienced similar sizing discrepancies in the past so I'm interested to know how you and Bioo interpret these results.

Adam, I used to using V1-V3, couldn't get the exactly pure one band peak either, much more impure than the one I showed here. But I used Pippin Prep to pick the correct size libraries, which help us get decent MiSeq data. It's the first time I used V4, I'm following Brad's suggestion to see what's gonna happen. But I'm working on human microbiome, other than gut microbiome, I can't be sure if my samples contain bacteria. Anyway, I will update this thread if those libraries get sequenced.
Good luck!


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