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Dear All,
I followed the MEDUSA protocol to filter out both not properly paired, low quality mapping and non-unique sequences from my alignment files.
For example one mC sample started with 100 milions reads. 80 % mapped, 70 % of them properly mapped with high quility (mapQ>40).
The problem arises when I filter out for non-unique reads. Roughly 90 % are discarded leading to a final number of 3-5 milions of reads.
All my mC samples behave in the same way.
Maybe the DNA starting material was not properly quantified (2-3 ng instead of 5 ng were used for the generation of the libraries).
We didn't observe the same problem for the Input DNA ( correctly quantified) and for 2 samples out of 4 for 5-hydroxy-mC.
The high number of non-unique reads could be due to a technical problem or a biological problem? Have you ever experienced a similar problem?
How do you think I should proceed with the analysis? Is it absolutely necessary to remove non-unique reads?
Is the first time I deal with this kind of analysis I would like to undestand which is the best approach to follow.
Thank you very much for your time,
Paolo
I followed the MEDUSA protocol to filter out both not properly paired, low quality mapping and non-unique sequences from my alignment files.
For example one mC sample started with 100 milions reads. 80 % mapped, 70 % of them properly mapped with high quility (mapQ>40).
The problem arises when I filter out for non-unique reads. Roughly 90 % are discarded leading to a final number of 3-5 milions of reads.
All my mC samples behave in the same way.
Maybe the DNA starting material was not properly quantified (2-3 ng instead of 5 ng were used for the generation of the libraries).
We didn't observe the same problem for the Input DNA ( correctly quantified) and for 2 samples out of 4 for 5-hydroxy-mC.
The high number of non-unique reads could be due to a technical problem or a biological problem? Have you ever experienced a similar problem?
How do you think I should proceed with the analysis? Is it absolutely necessary to remove non-unique reads?
Is the first time I deal with this kind of analysis I would like to undestand which is the best approach to follow.
Thank you very much for your time,
Paolo