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Old 04-16-2018, 04:50 AM   #4
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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Quote:
Originally Posted by AmitChaurasia View Post
Hi Kinman,

Thanks for your reply, I have taken random samples to check with Bioanalyzer and no short peaks were found. Also fastq files were checked to have 301 base length only.

So this can be ruled out.

Best,
The length of the reads in your .fastq file likely does not include low-quality base clipping. The MiSeq will happily call bases on background noise. So the length of the reads is not diagnostic for this purpose.

Your random samples that were run on the bioanalyzer -- did they include any of the ~dozen samples that contributed >2% of reads? Those would be the likely culprits for providing large amounts of primer dimers.

Even if these did not show primer dimers your samples may have included large amounts of primer-dimers annealed to full length library molecules. To detect these you would need to use some sort of denaturing electrophoretic size analysis. This could be denaturing PAGE, or if you prefer the Agilient Bioanalyzer, try:

denature your samples for 3 minutes at 95oC and then "snap cool" them in an ice-water bath. Then run them on a denaturing agilent chip -- we use the RNA pico chip for this purpose. Note that during the heat denaturation step you need some method to prevent your sample from just evaporating -- use of a heated lid thermal cycler is good for this purpose.

--
Phillip
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