Hi Guys,
I am trying to come up with my own amplification and sequencing primers for 16s V1V3 region and I would like to get help with primer design.
I think Caporaso primer for V4 region is a good start to do modifications on. I want to understand how the primer pad was designed. In his paper, the explanation for pad region is "The amplification and sequencing primers additionally contain a new pad region to avoid primer-dimer formation with the modified adapter. " But I still don't know how to adapt this for other variable regions. Any ideas?
515F (forward primer) PCR primer sequence:
Field number (space-delimited), description:
1, 5' Illumina adapter
2, Forward primer pad
3, Forward primer linker
4, Forward primer
AATGATACGGCGACCACCGAGATCTACAC TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
806R (reverse primer) PCR primer sequence (each sequence contains different barcode):
2168 GoLay barcoded reverse PCR primers. Each primer is followed by a barcode identifier
generated specifically for this set of primers.
Field number (space-delimited), description:
1, Reverse complement of 3′ Illumina adapter
2, Golay barcode
3, Reverse primer pad
4, Reverse primer linker
5, Reverse primer
CAAGCAGAAGACGGCATACGAGAT TCCCTTGTCTCC AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT 806rcbc0
Read 1: (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA)
Index: (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT)
Read 2: (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT)
I am trying to come up with my own amplification and sequencing primers for 16s V1V3 region and I would like to get help with primer design.
I think Caporaso primer for V4 region is a good start to do modifications on. I want to understand how the primer pad was designed. In his paper, the explanation for pad region is "The amplification and sequencing primers additionally contain a new pad region to avoid primer-dimer formation with the modified adapter. " But I still don't know how to adapt this for other variable regions. Any ideas?
515F (forward primer) PCR primer sequence:
Field number (space-delimited), description:
1, 5' Illumina adapter
2, Forward primer pad
3, Forward primer linker
4, Forward primer
AATGATACGGCGACCACCGAGATCTACAC TATGGTAATT GT GTGCCAGCMGCCGCGGTAA
806R (reverse primer) PCR primer sequence (each sequence contains different barcode):
2168 GoLay barcoded reverse PCR primers. Each primer is followed by a barcode identifier
generated specifically for this set of primers.
Field number (space-delimited), description:
1, Reverse complement of 3′ Illumina adapter
2, Golay barcode
3, Reverse primer pad
4, Reverse primer linker
5, Reverse primer
CAAGCAGAAGACGGCATACGAGAT TCCCTTGTCTCC AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT 806rcbc0
Read 1: (TATGGTAATTGTGTGCCAGCMGCCGCGGTAA)
Index: (ATTAGAWACCCBDGTAGTCCGGCTGACTGACT)
Read 2: (AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT)
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