Alm lab Protocol for processing overlapping 16S rRNA reads from a MiSeq run
Experimental design
The specifics of the sequencing set-up and molecular construct will determine exactly how the data needs to be sequenced and processed. There are a few different designs that the Alm lab has set up:
1.) Multiplexing different samples together to be sequenced in one lane of Illumina, marking each unique sample with a unique barcode on the reverse primer of step 2 that is read during the indexing read. This is common for up to 96 samples (or 105 including additional barcodes that are not in the 96-well format). The sequencing should be done, not with the standard Illumina indexing primer, but with the reverse complement of the 2nd read sequencing primer. This is a custom barcode that should be included in the
2.) Multiplexing multiple different plates of samples together using a barcode located 5' to the primer used in genome amplification (typically U515) and a reverse barcode that is read during the indexing read.
De-multiplexing