PacBio ZMW Loading For Productivity

[juckdnarocks]

Hi all,
I recently constructed a 10k library and sequenced on RS II platform with P4 C2 chemistry, and got an abnormal result of ZMW loading for productivity with P0 74.22%, P1 13.48%, P2 12.3% and mean read length 1,324 bp, mean read quality 0.81.
I refered to the help of smrt portal, which says
"Productivity 0 (%): Percentage of ZMWs that are empty, with no polymerase.
Productivity 1 (%): Percentage of ZMWs that are productive and sequencing.
Productivity 2 (%): Percentage of ZMWs that are not P0 (empty) or P1 (productive). This may occur for a variety of reasons and the sequence data is not usable."

But I still don't know the cause of the abnormal ZMW loading result.
Does P0 means that ZMW has no polymerase loading or no template loading or contaminants deactivate the polymerase？
Anther question is exactly what reasons for P2 occurring ?

Could someone help to explain ? Thx very much.

[GenoMax]

Loading of the ZMW's follows a poisson distribution. It generally results in ~40% of the wells loading with a single molecule (as you probably saw in the PacBio training materials). Basic idea is to maximize P1 while reducing P0 (no molecules/no signal) and P2 (more than one set of signal i.e. mixed sequence) as much as possible.

You should consult with your local PacBio Field Application Scientist on figuring out how to increase the P1%. Though I do not work in the lab it is my understanding that this is somewhat an imprecise process that may require some trial and error.

[tacana]

From the information that you show, to me it looks like your cell was a bit underloaded. A P0 means that the instrument is not able to detect any signal on that ZMW. There are several reasons for this to occur, just like you noted this can happen if there is s no polymerase loaded or no template loaded or contaminants deactivate the polymerase. If contaminants are not an issue, increasing the on-plate concentration should improve the % P1.

Good luck!

[juckdnarocks]

From the information that you show, to me it looks like your cell was a bit underloaded. A P0 means that the instrument is not able to detect any signal on that ZMW. There are several reasons for this to occur, just like you noted this can happen if there is s no polymerase loaded or no template loaded or contaminants deactivate the polymerase. If contaminants are not an issue, increasing the on-plate concentration should improve the % P1.

Good luck!

Thanks very much. But how to determine which contaminant deactivates the polymerase is the problem. Is possible to detect it? I am not familiar with experiment.

[tacana]

It is difficult to figure out which contaminants affects the Pacbio polymerase since many of these contaminants can be unique to an organism, growth conditions, DNA extraction method, etc.
However, one way that can give you a hint that it is a contaminant issue is the distribution of ZMWs. For example, if you see a ZMW distribution pattern in which you have a percentage of P0 and P>1, and a low % of P1, then you should suspect contamination issues.

[MatDeclercq]

As tacana mentioned, a rather high P0, low P1 and moderate P2 is quite often a signature of some contaminating compounds adhered to the SMRTbells.
In your case p1 and p2 are almost similar, which makes it a bit borderline to tell.
On the other hand, quality scores of 0.81 and especially mean read lengths of 1.3Kb are extremely poor for a P4 polymerase (if by mean RL you're talking about the mean polymerase RL and not mean Read of Insert size).
In this case I would say you have a contaminant problem. Do you have any information on the DNA extraction method?

Either way, a second run with double or triple the loading concentration will be the only way to be absolutely sure.
If this does not improve p1 drastically, your last hope is a MagBead clean-up (available on samplenet) to try and get rid of the contaminant. But no guarantees there whatsoever...

Good luck!

[raibiotech]

hi,

@ MatDeclercq
As you mentioned :

Either way, a second run with double or triple the loading concentration will be the only way to be absolutely sure.

But as per Pacbio manual : They have fixed conc. on plate =0.015nM..

How we can change that?
Please suggest...