Usually during the design of fusion primers for amplicon sequencing in 454 GS FLX Titanium using Lib-L kit, Adapter A is attached with the forward amplification primer and Adapter B with the reverse primer (as per Roche manual). In using Lib-L kit, sequencing is unidirectional and is from Adapter A only. Will the sequencing data be affected if we change the attachment positions of the adapters (Adapter A attached with Reverse primer and Adapter B with Forward primer)? If there are affects, how?
No, you may attach the A adapter to either the forward or reverse primer. Be sure to add the tag (e.g. barcode sequence) to the reverse fusion primer for sorting. Downstream processing will, of course, require reverse complementation after your initial quality processing, unless you are directly classifying your sequences without clustering (e.g. RDP Classifier) or unless you are using a protein translation tool that corrects this.
I agree with Christopher. Be careful with downstream analzses and DO NOT forget RC your sequences if you will sequence form reverse primer.
Thank you for the answers. We have already designed the fusion primers. The MID is added downstream of Adapter A and upstream of the Reverse primer.
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