This is an adapted protocol with fewer steps and a bit more flexibility than that published, optimized for high-throughput studies with many samples, and samples with lower DNA yield. Main changes are:

1) The template-tagging primers and PNAs are added at higher concentration and smaller volume to save more reaction volume for DNA.

2) The template tagging primers are at twice the final reaction concentration published. Because complex samples have many off-target binding sites, this makes it a little more likely that all true templates are tagged, and extra primer is purified away before PCR.   

3) For 16S protocols, tagging with the forward primer occurs first, as opposed to tagging first with the reverse primer as in the publication. This is because the forward primers we use (338F, 515F),  have a higher annealing temperature than the reverse primer (806R). By tagging first at a higher annealing temperature, off-target amplification in the initial tagging events is less likely. If you are not doing a 16S amplicon, start your tagging with the primer that has the higher melting temperature.

4) We sometimes do linear amplification with the forward primer so that template molecules can be enriched in samples with very low DNA.  Take note: linear amplification means that the same original template molecule can be tagged more than once, so this means molecule tags are less effective for correcting PCR sampling bias. However, in linear amplification (as opposed to PCR), all copies are being made from the original template, and so errors don't accumulate as they would if you were making copies of copies of copies etc. Thus, despite doing linear amplification, you can still use molecule tags to correct error down to the very first tagging event (it is better than not using molecule tags). If you are getting product and your samples do not require linear amplification, do not do it.

5) We do not bead clean the reaction after the first tagging event. The polymerase and dNTPs, etc., are all still fresh from the minimal cycling, so we simply take the PCR plate off the machine and put it on ice, add the second template-tagging primer to each reaction, mix well, and put it back on the machine for one more cycle to finish the tagging.

6) We use a bead:DNA ratio of 0.6:1 for all steps and purifications. (Also, we use home-made beads, which are >50 times cheaper than Agencourt beads. See our other online lab protocol). We find that if some beads from the tagging step go into the PCR step, this is fine. They are inert and you can purify them out later.

7) For large projects with deep multiplexing, we don't bead clean the PCR of each sample independently, and we don't quantify the product from every PCR reaction in order to normalize the library. We simply mix 2uL from every sample into a single pool, bead purify this twice in a row (bead:DNA ratio 0.6:1) and then quantify and sequence this library.


Note cycling is exactly the same for the 338F and 515F primers

Step 1 in 25uL:
    5uL           Kapa Enhancer
    5uL           Kapa Buffer A
    0.4uL        5.0 uM forward tagging stock
    0.375uL    mixed PNAs (1:1 mix of 100uM pPNA and 100uM mPNA stocks)
    0.5uL        Kapa dNTPs
    0.25uL      Kapa Robust Taq
    13uL         DNA (or DNA + water)

SUM 24.5uL

Mix well and cycle at:
   45 sec at 95C followed by 1 to 30 cycles (depending on linear amplification needed) of:
   95C x 15 sec
   78C for 5 sec (PNA annealing)
   60C x 30 sec (515F or 338F annealing. Mean melting is >60C )
   72C x 30 sec
   finish at 4C

Remove reaction from 4C and add, while on ice:
1.6uL        water
   0.4uL        5.0 uM reverse tagging stock
SUM 26.5
Mix well and do 1 cycle of:
   95C x 1 min
   78C for 5 sec (PNA annealing)
   50C x 60 sec (806R annealing. Mean melting is ~50C)
   72C x 60 sec

Bead clean using bead:DNA ratio of 0.6:1 and elute in 21 uL

Step 2 in 50uL:
   25uL        Kapa HiFi HotStart ReadyMix
   0.75uL     mixed PNAs (1:1 mix of 100uM pPNA and 100uM mPNA stocks)
   5uL          index primer
   20uL        DNA from the forward templatetagging step (dont worry if some beads come along)

Cycle at:
   45 sec at 95C followed by
   35 cycles of:
   95C x 15 sec,
   78C for 5 sec (PNA annealing)
   60C x 30 sec (index primer annealing)
   72C x 30 sec
   Finish at 4C

Sample 338F-806R samples after PCR step (but before any bead purification)

Sample 338F-806R library made by pooling 2uL from 192 samples, bead cleaned twice and ready for sequencing (note disappearance of dimers)