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  • isalarubi
    replied
    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!!

    Leave a comment:


  • newmoon
    replied
    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.

    Leave a comment:


  • newmoon
    replied
    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.

    Leave a comment:


  • isalarubi
    replied
    Dear newmoon,

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

    Thank you!!

    Isabel
    Attached Files

    Leave a comment:


  • newmoon
    replied
    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

    Leave a comment:


  • isalarubi
    replied
    Dear Newmoon,

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

    Thank you!
    Attached Files

    Leave a comment:


  • newmoon
    replied
    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.

    Leave a comment:


  • bunce
    replied
    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!

    Leave a comment:


  • isalarubi
    replied
    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

    Leave a comment:


  • bunce
    replied
    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

    Leave a comment:


  • isalarubi
    replied
    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

    Leave a comment:


  • ajthomas
    replied
    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.

    Leave a comment:


  • isalarubi
    started a topic Problems with 16S sequencing 454Jr

    Problems with 16S sequencing 454Jr

    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

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