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Old 04-17-2018, 09:13 AM   #7
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
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)
Those are just 16S sequences. Since FastQC only appears to be looking at the first 50 bases, I don't think it is a good assessment of the presence of primer dimers in your libraries. You could follow Genomax's advice. But it would be good to see the FastQC of the reverse read. If it is the reverse complement of that sequence then you either have a very short v-loop in the species you are sequencing, or you do have primer-dimers.

Do you have a service contract? If so, just ask Illumina for reagents to do a phiX run. This would show if your MiSeq had deteriorated severely from its normal specifications.

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Phillip
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