Hello All,
I have been scouring the internet to try to find the answer to my question, but to no avail. We are attempting to sequence multiple barcoded 16s paired-end amplicons on the Miseq and to get around the low-diversity issue we plan to stagger the sequencing start position for our runs by 0-5bp (incorporated into our amplicon oligos).
The question that I have concerns the cluster identification. If the read 1 sequence has the first 4bp at a ratio of 1:1:1:1 for A:T:G:C and the identification of clusters is good, are the values obtained here used for read 2 as well? Or does the same process occur on read 2 with cluster identification... I would assume not since the location on the flow-cell determines the clusters, and doing this twice may give two completely different cluster location "pictures".
Any insight here would be greatly appreciated! Thanks!
I have been scouring the internet to try to find the answer to my question, but to no avail. We are attempting to sequence multiple barcoded 16s paired-end amplicons on the Miseq and to get around the low-diversity issue we plan to stagger the sequencing start position for our runs by 0-5bp (incorporated into our amplicon oligos).
The question that I have concerns the cluster identification. If the read 1 sequence has the first 4bp at a ratio of 1:1:1:1 for A:T:G:C and the identification of clusters is good, are the values obtained here used for read 2 as well? Or does the same process occur on read 2 with cluster identification... I would assume not since the location on the flow-cell determines the clusters, and doing this twice may give two completely different cluster location "pictures".
Any insight here would be greatly appreciated! Thanks!
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