Problems with 16S sequencing 454Jr

[isalarubi]

Hi everybody!

We're new with the 454 Jr, and in our first run of 16S to determine the stool microbiota we found some problems.

We've had rawWells,304998 and then only keyPassWells,12489. When we see the color system, all are in pink as no key.

Anybody knows what is happening?

Thank you

[ajthomas]

Are the key pass wells all control beads? If so, I would guess that you grabbed the wrong tube when you annealed the sequencing primer and used the enrichment primer instead.

[isalarubi]

Thank you ajthomas, but we are quite sure that we did not use the primers in the wrong way.

We do not understand the program very well yet. Probably our answer would be in the files, but we do not know whre we have to see

[bunce]

Hi isalarubi,
Are you using LibA or LibL? - In our experience with amplicon sequencing on the Gs-jnr you want to aim for only 180,000-ish raw wells. The lack of complexity in the amplicon seems to cause the plate to flare. My advice is to be very careful about your bead:template ratio and load conservatively (i.e about 4% which is just below the line on your bead counter). If your control bead are not sequencing well it is often because you are overloaded. Cheers Mike

[isalarubi]

Thank you Mike,

we are using Lib L. Definitely we need to be more conservative with the bead:template ratio, so that we were working with a higher one as in the training told us, but in our case it doesn't work.

Thank you so much!!

Isabel

[bunce]

For Lib-L with amplicons drop the emPCR primer down to a quarter and aim to load 180,000-ish beads. The platform is very sensitive to bead:template ratios - my advice if you are getting going is to use a qPCR assay to dial-in your ratio…. just because the bead enrichment is "in the window" is no guarantee that the ratio was 'correct'. good luck!

[newmoon]

would you post the filtermetrics summary so we can look at the whole run including the read length and control DNA metrics?

You were overloading the JR, but I don't think that's the reason the key pass well was so low.

[isalarubi]

Dear Newmoon,

here you have the files. I hope you can give us some information from them.

Thank you!

[newmoon]

hi isalarubi,

this is not exactly what I want to see.
In the GS Run Browser, you can find different tags for wells, readlength, control DNA...
I have an example of filtermetrics from a Flx run / 2 region PTP here. If you can find these numbers for your run, I might have a clue about the problem.
(I have hard time to upload the file, too big. I past it here.)


Filter Metrics, GACT
GACT (Library) Region
1 2 Total
Raw Wells 945,367 955,708 1,901,075
Key Pass Wells 897,232 900,375 1,797,607

Failed Dot 23,309 10,154 33,463
Mixed 239,743 146,537 386,280
Short Quality 161,548 147,548 309,096
Short Primer 9,296 2,925 12,221

Passed Filter Wells 457,269 587,106 1,044,375

% Dot + Mixed 29.32 17.4 23.35
% Short 19.04 16.71 17.87
% Passed Filter 50.96 65.21 58.1



Read Length and Quality Metrics, Read Length, GACT
GACT (Library) Region
1 2 Total
Raw Wells 945,367 955,708 1,901,075
Key Pass Wells 897,232 900,375 1,797,607
Passed Filter Wells 457,269 587,106 1,044,375
Total Bases 149,917,353 208,858,977 358,776,330

Length Average 327.85 355.74 343.53
Length Std Deviation 105.42 101.17
Longest Reads Length 1,193 1,196 1,196
Shortest Reads Length 40 40 40
Median Reads Length 349 377 365



Control DNA Metrics
Region
1 2 Total
Raw Wells 945,367 955,708 1,901,075
All ControlDNA Wells 17,340 20,625 37,965
Average
% Match 200 bp, 100% 80.4 77.64 78.9
≥98% 92.68 91.13 91.84
≥95% 94.94 93.64 94.23

[isalarubi]

Dear newmoon,

sorry, for the wrong file, I think this new one is the good one.

Thank you!!

Isabel

[newmoon]

I only see the filter metrics. It was bad with the key pass wells being so low.

what about the read length, is median read length around 350bp?

and what about control DNA metrics, is %match 95% above 85%? If the control DNA was good, that means the run was good, but your beads had problem.If the control DNA has 95% match below 75%, I would think the run itself had problem, may not the beads problem.

The only time we had low key pass wells was when our machine had fluidic issue which leaked during the flow. If you know how to look at the images, you may find a clue.

Have you sent a run report to 454 tech support for a diagnosis? They should be able to tell you if it's machine problem or beads problem.

[newmoon]

I don't know why the reply I posted yesterday didn't show up here.

anyway, I would suggest to look at the control DNA metrics which you didn't post in the last post. If the control %match 95% was above 80, the run itself was good, which means the beads had problem. If the control %match 95% is below 75, the run had problem, which could be the reagents or the machine had problem.

have you sent a run report to 454 tech support? They should be able to tell you if the machine or the beads had problem.

[isalarubi]

Thanks a lot NEwmoon,

we are in touch with the technical support from Roche, and well, they have said that the machine is ok, and that it could be only an isolated event, and I hope so.

We are in the middle of a new run, and I hope it will be ok.

Fingers crossed!!