Hi All,
I'm hoping one or more of you can help to clarify some issues for me, and/or confirm whether I understand things correctly. There is extensive and detailed information about the adaptor sequences, primer sequences, etc. for other platforms available on this site, but I haven't found anything nearly so clear for 454. And it seems to be there is a fairly complicated situation for 454.
As I understand it, 454 FLX (not Titanium) used a single set of "core" adaptor/PCR primer sequences for all libraries, including amplicon libraries, and these are as follows:
FLX Primer A: 5’-GCC TCC CTC GCG CCA-3’
FLX Primer B: 5’-GCC TTG CCA GCC CGC-3’
Then, 454 FLX Titanium employed a completely different set of adaptor/PCR primer sequences for "standard" libraries, as follows:
Titanium Primer A: 5’-CCA TCT CAT CCC TGC GTG TC-3’
Titanium Primer B: 5’-CCT ATC CCC TGT GTG CCT TG-3’
When Roche later released protocols for Titanium sequencing of amplicon libraries, they essentially used slightly modified (extended at the 5'-ends) "FLX" adaptor sequences. However, apparently because some users wanted to do just unidirectional sequencing of their amplicon libraries, they later released protocols describing Titanium "Lib-L" - compatible Fusion Primers.
From their website, it seems they currently supply two types of Titanium emPCR and sequencing kits -- "Lib-L" or "Lib-A". I assume "Lib-L" stands for "library" or something along those lines, and "Lib-A" denotes "Amplicon". I also assume that Titanium "Lib-L" emPCR and sequencing kits essentially use the Titanium sequences listed above, while the Titanium "Lib-A" emPCR and sequencing kits use the (modified/extended) 454 FLX sequences, based on those above.
Were there ever "Lib-A" and "Lib-L" versions of the 454 FLX kits? Or do these terms only refer to the Titanium range?
How does unidirectional vs bidirectional sequencing work on the 454 platform, and why does one need special emPCR kits to enable each? Surely one can use either A or B primer as the sequencing primer?
Any insight into this would be greatly appreciated.
I'm hoping one or more of you can help to clarify some issues for me, and/or confirm whether I understand things correctly. There is extensive and detailed information about the adaptor sequences, primer sequences, etc. for other platforms available on this site, but I haven't found anything nearly so clear for 454. And it seems to be there is a fairly complicated situation for 454.
As I understand it, 454 FLX (not Titanium) used a single set of "core" adaptor/PCR primer sequences for all libraries, including amplicon libraries, and these are as follows:
FLX Primer A: 5’-GCC TCC CTC GCG CCA-3’
FLX Primer B: 5’-GCC TTG CCA GCC CGC-3’
Then, 454 FLX Titanium employed a completely different set of adaptor/PCR primer sequences for "standard" libraries, as follows:
Titanium Primer A: 5’-CCA TCT CAT CCC TGC GTG TC-3’
Titanium Primer B: 5’-CCT ATC CCC TGT GTG CCT TG-3’
When Roche later released protocols for Titanium sequencing of amplicon libraries, they essentially used slightly modified (extended at the 5'-ends) "FLX" adaptor sequences. However, apparently because some users wanted to do just unidirectional sequencing of their amplicon libraries, they later released protocols describing Titanium "Lib-L" - compatible Fusion Primers.
From their website, it seems they currently supply two types of Titanium emPCR and sequencing kits -- "Lib-L" or "Lib-A". I assume "Lib-L" stands for "library" or something along those lines, and "Lib-A" denotes "Amplicon". I also assume that Titanium "Lib-L" emPCR and sequencing kits essentially use the Titanium sequences listed above, while the Titanium "Lib-A" emPCR and sequencing kits use the (modified/extended) 454 FLX sequences, based on those above.
Were there ever "Lib-A" and "Lib-L" versions of the 454 FLX kits? Or do these terms only refer to the Titanium range?
How does unidirectional vs bidirectional sequencing work on the 454 platform, and why does one need special emPCR kits to enable each? Surely one can use either A or B primer as the sequencing primer?
Any insight into this would be greatly appreciated.
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