FLX vs Titanium, Lib-L vs Lib-A, Amplicon Libraries = confusion

[SeqTruth]

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.

[Marzanna]

Hi,

The Lib-L stands for Library created by Ligation and Lib-A for Library created by Amplification (with respective primers).

FLX had three different kits for ePCR but not Lib-L and Lib-A which are only for Titanium.

The kit Lib-A contains two different types of beads (A and B). Library fragments are attached to the beads with either A or B adapter and the other one is used for sequencing (direction towards the bead). So the beads have to fit to the direction one want to use for sequencing. Hence for bidirectional sequencing one uses both types of beads.
For unidirectional amplicon sequencing one can prepare ePCR with either A or B beads from Lib-A kit (using two kits as the kit has half A and half B beads) or with Lib-L kit if the primers were designed for it (only A direction is possible).

Cheers,
Marzanna

[SeqTruth]

Thanks Marzanna! That has cleared up a number of points of confusion for me

[eladSeq]

Using the Lib-A emPCR kit, we are having an issue where we observe good enrichment with the "B" beads, but no enrichment using the "A" beads. Since the amplicons each have an A and B adapter sequence why would one work but not the other? Should we try higher molecules/beads, I think 1,2 and 4 was tried.

Also,
One other question regarding, regarding the A and B capture beads. If the A beads bind the A adapter sequence, then the sequencing primer must bind the B adapter, and read direction will be from B->A (the reverse complement). And similarly if the B capture beads bind the B adapter then those beads will provide reads in the A->B direction. Is this correct?

[MissDNA]

We´ve never used Lib-A kit but soon we will.
I would like to know if you guys titrate separatedly beads A and B, and for the sequencing emPCR when the beads are combined: emPCR, recorery or only prior to loading the plate. Our project it will be a 16-lane plate, so we will be using SV. I believe we will have enough beads after titration to load the plate.