Hi all!
I have some 16S sequences amplified with the universal primers 341F: CCTACGGGNGGCWGCAG & 805R: GACTACHVGGGTATCTAATCC. I have checked the sequences and primers are present in all sequences, yet, they do not have ambiguous bases. For example, for 341-F instead of having: "CCTACGGGN" there is, "CCTACGGGT".
I want to use DADA2 to analyze these sequences, but I have read the following: "DADA2's chimera removal step requires primers to have been removed, as otherwise the ambiguous nucleotides in most primer sets cause large numbers of false-positive chimeras to be identified". But, sequences do not have ambiguous nucleotides...
My question is, does keeping primers (without ambiguous nucleotides) could affect 16s subsequent analysis?
And if so, how would it affect it?
Any help/insight will be appreciated. Thanks in advance!
- My question is, does keeping primers (without ambiguous nucleotides) could affect 16s subsequent analysis?
>> YES!
- And if so, how would it affect it?
>> This question has been asked several times on research gate, please search if you are interested in knowing the reason behind it.
Hi, They can impact your analasis. I would remove the primers from your reads informatically, or alternatively, if you are sequencing libraries in the future use custom sequencing primers that correspond to your PCR primers, that way the resulting sequences don't include the primes.
Frank
Thanks for your replies. Abhijeet Singh Could you please, share any link with the discussion about 16s primers effect in downstream analysis?
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