(MW 6/26/20) Amplicons were generated by Jenny using a custom (non-standard) 2-step PCR protocol, summarized below:
1st PCR = 16S primers (27F and 1492R) = WITH 5' block, WITHOUT universal sequences, WITHOUT barcodes
2nd PCR = Asymmetrical barcoded primers = WITHOUT 5' block
(Combinatorial F + R barcode pairings -- See list in table above)
The barcoded amplicons were then multiplexed into a single tube for bulk library prep using a nonbarcoded SMRT Bell adapter. Therefore, the assymetrical sample barcodes appear within the insert rather than the typical location within the SMRT bell. We anticipate Jenny having a custom script to demux this data after we perform CCS analysis.
1st PCR = 16S primers (27F and 1492R) = WITH 5' block, WITHOUT universal sequences, WITHOUT barcodes
2nd PCR = Asymmetrical barcoded primers = WITHOUT 5' block
(Combinatorial F + R barcode pairings -- See list in table above)
The barcoded amplicons were then multiplexed into a single tube for bulk library prep using a nonbarcoded SMRT Bell adapter. Therefore, the assymetrical sample barcodes appear within the insert rather than the typical location within the SMRT bell. We anticipate Jenny having a custom script to demux this data after we perform CCS analysis.