We are trying to sequence (illumina - miseq) bacterial population from mouse feces. We are amplifiying the V4 region, and the problem is that instead of having only one band/peak corresponding to about 400bp for V4 region, we have this band plus a smir on top of it, of higher molecular weight. I was wondering if this is an artifact of something else which could be amplified, like the mouse genomic DNA for example.
I was wondering about the V4 range of size variation that could be found in mouse feces(is it just a few base pair, or hundreds of it).
We are using Patrick Schloss protocol, using promers 515F and 806R targeting v4 region of 16S
515F AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TATGGTAATT GT GTGCCAGCMGCCGCGGTAA (red is targeting V4 region, blue is sequence to anneale to flow cell, green is pad, X index)
806R: CAAGCAGAAGACGGCATACGAGAT XXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT
Could it be because of mitochondrial genes ? I wonder how variable in size is 16S?
I was wondering about the V4 range of size variation that could be found in mouse feces(is it just a few base pair, or hundreds of it).
We are using Patrick Schloss protocol, using promers 515F and 806R targeting v4 region of 16S
515F AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TATGGTAATT GT GTGCCAGCMGCCGCGGTAA (red is targeting V4 region, blue is sequence to anneale to flow cell, green is pad, X index)
806R: CAAGCAGAAGACGGCATACGAGAT XXXXXXXX AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT
Could it be because of mitochondrial genes ? I wonder how variable in size is 16S?
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