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Old 01-14-2011, 03:48 AM   #1
Phimes
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Default Titanium amplicon fusion primer confusion

Hello all, I'm very new to this and I may be asking a silly question but I'm generally a bit confused about the adapter molecules and the "key" sequence on the A and B primers. Right, so basically i would like to perform unidirectional sequencing from the A fusion primer on bacterial 16S amplicons using the Lib-A chemistry on the Roche Titanium platform. I have been told that you can remove the 4 bp key from the B adapter molecule on the fusion primer to increase the read length of the sequence of interest. So basically, do I order the A fusion primer (|A adapter sequence|key|forward primer|) and the B fusion primer (|B adapter sequence|reverse primer|) and use the B beads from the kit, then sequence from the A fusion primer towards the bead? The sequencing center will be doing the library prep and sequencing etc, but i just wanted to 1) make sure I understood what was going on, and 2) make sure i order the correct fusion primers!

Cheers everyone,

Dave
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Old 01-14-2011, 04:24 AM   #2
Phimes
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Sorry, all sorted now. I was being a fool!

Dave
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Old 01-17-2011, 06:12 AM   #3
flxlex
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In that case, please answer your own question for the interested readers!
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Old 01-17-2011, 06:53 AM   #4
kmcarr
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If you are only interested in unidirectional sequencing of your amplicon you can use the Lib-L kit instead of the Lib-A kit. The primers you design will need to be specific for the Lib-L kit. Roche has an Application Brief for making Amplicon libraries for use with the Lib-L kit (attached). The advantages for using Lib-L for unidirectional amplicon sequencing are 1) you're not paying for components which you are going to throw away (the "B" components) and 2) you get 70 million capture beads of one type.
Attached Files
File Type: pdf APP001-2009_Lib-LunidirectionalAmplicons.pdf (231.6 KB, 88 views)
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16s rrna gene, amplicons, fusion primers, key, lib-a

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