Ligating titanium adapters to 16S amplicons...

[dc3000]

Has anyone examined 16S diversity (of a single DNA extraction) using an Adapter-MID primer set and compared it to the amplicon generated using otherwise identical unbarcoded, adapterless primers (with Adapter-MID ligated to the amplicon)? It seems that the latter would be an easy way to reduce the headache of PCR-optimization for tricky primers and reduce potential barcode bias.

To perform the ligation, would it be best to use a protocol such as this by Zheng et al. 2010 and use the Lib-A kit?

Adapter ligation was conducted in a 25 µl reaction with 1× slow ligation buffer, 0.4 pmol of each of the A4 and B adapter (Invitrogen, USA) and T4 DNA Ligase 180 U (Enzymatics, MA, USA). We used the FLX Titanium adapter A4 (GS multiplex identifier number 4) because this adapter had performed well in previous experiments in our lab. The ligation reaction was incubated at 22°C over night, purified by AMPure beads (17.5 µl, 70% volume) and eluted in 25 µl TE. The eluted DNA was treated with 8 U Bst DNA polymerase Large Fragment (New England Biolabs), to fill in the 3′-junction nick between the adapter and sample DNA in a 30 µl reaction with 1× fill-in buffer, 30 µM dNTP and incubated at 37°C for 20 min. The double-stranded DNA library was purified with AMPure beads (21 µl, 70% volume) and eluted in 30 µl TE.

[RCJK]

Hi dc,

If you can, check out the Application Brief APP No. 001-2010, there is a section on preparing ligated amplicon libraries. Here's part of it:

To create a ligated Amplicon library, follow the GS FLX Titanium Amplicon Library Method Manual using oligonucleotides that contain the gene specific sequence only (DO NOT include the adaptor sequence) for the loci. After the solution phase PCR reaction has completed and size selection has been performed, follow the GS FLX Titanium Rapid Library Method Manual with the following exceptions:
1. Begin at Section 3.2 End Repair (skip Section 3.1 entirely)
2. At Section 3.2.2:
Start with 500 ng of quantified amplicon material prepared from GS FLX Titanium Amplicon Library Preparation or 500 ng of any dsDNA product as the DNA sample. Bring sample volume up to 16 μl with 1xTE.
3. Continue with the remainder of the protocol as written
NOTE: Additional deviations from the outlined protocol above may be required depending on the amplicon design (in particular, the length of the amplicons):
o Long(>1000bp)amplicons: Nebulization should be considered for full length, double-stranded sequencing.
o Short(<400bp)amplicons: An increase in SPRI bead concentration (without Sizing Solution) to 1.8x may be required, however this will depend on the specific amplicon length.

[dc3000]

Thanks RCJK.

Since I only need to perform unidirectional sequencing, is it necessary to use both 'A' and 'B' adapters?

Is there any trick to ensure proper orientation of the adapter during ligation?

[pmiguel]

Yes it is necessary.

Remember that prior to emulsion PCR you titrate your library such that most of the beads occupy a microreactor with 1 or 0 library molecules. The GS-FLX is not sensitive enough to detect the signal from a single template molecule. To produce millions of (identical) template molecules PCR is used. PCR can only produce the necessary exponential amplification if it has a priming site at each of the amplicon (library molecule).

--
Phillip