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Old 04-17-2018, 02:23 AM   #5
AmitChaurasia
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Location: Delhi, INDIA

Join Date: Jan 2013
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Quote:
Originally Posted by pmiguel View Post
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
Thanks a lot Phillip for your valuable inputs.

I have gone through your suggestions and looked into the data in greater details wrt possiblity of primer-dimer or adapter dimers. I have checked the fastq files with fastqc software to look for over-represented sequences but could not find the adapter-dimers or primer dimer sequences in that. SO i believe I can rule out the contamination of these dimers. On the other hand I have also checked out for over-represented samples if any of them have BA traces, I found none of them. I am planning to get BA profile for a few over-represented libraries done to get 100% sure that if there is any contamination in them (is it necessary to do BA on RNA-chip?)

I am attaching fastqc screenshot for your reference (same sequences are over-represented in all fastq files)
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