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QA/QC Policies|University of Wyoming | Macromolecular Analysis Core

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Macromolecular Analysis Core
Sequencing Core
AgC 5024B
766-2922
Macro Core
Physical Sciences 108
766-3688
UW Campus
Phone: 307-766-2922
Email: uwmac@uwyo.edu
Macromolecular Analysis Core
Dept 3944
1000 E University Ave
Laramie, WY 82071
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Quality Assurance/Quality Control at the UW Macromolecular Analysis Core

 

We are genuinely interested in getting the best results attainable for our customers and we trust that they are genuinely interested in doing what it takes to get those best results.  We will work with you to optimize your preps and troubleshoot your results if you ask help.

We take great pride in our low error rate and go to great lengths to maintain it. These efforts include both proactive measures for the minimization of likely sources of errors and post-analysis detection of likely instances of mistakes. These measures are detailed here.

DNA Sequencing

Quality Assurance: Procedures used to minimize the likelihood of errors

  • All samples are checked for accurate labeling and similar customer errors before processing.

  • The customer enters the sample-specific information digitally at submission time so that sample names are not retyped.

  • Premixes are prepared with all reaction components except template and primer so that the same cocktail is present in all wells including positive control standards.

  • The final volume of the well is visually checked to verify the presence of the premix.

  • The volume of the plate is scanned visually after the purification step (Big Dye XTerminator) to verify consistency of volume in all wells before it is loaded onto the sequencer.

  • After the run, all results are scanned to check for symptoms of problems or of common failures (premix error, sequencer malfunction, salt effect, etc.).

  • If a standard fails or generates inappropriate sequence, the problem is identified and the reaction is rerun at no charge.

Quality Control: Measures implemented to detect problems after the fact

  • Each set of sequencing samples includes at least one standard with known purity and known performance as a positive control (and often several). If any standards do not sequence properly, it suggests a possible problem with the reaction or the instrument.

  • All sample sets are scanned to detect the presence of suspicious similarities between samples from adjacent capillaries. This detects any instance of capillary crosstalk.

  • Samples that fail under suspicious circumstances (single failures among larger sets of otherwise successful samples; large-scale failures from usually savvy customers; unrecognized failure modes) are repeated to test whether the sample was at fault or whether processing may have been erroneous.

  • Additional failed samples are repeated just for QC purposes, even if there is no indication of a problem in their handling. Typically, this is a spot-check of a few samples out of larger failed sets.

Please be aware that we provide a raw sequencing service; in other words, we will perform basic sequencing reactions (one template sequenced with one primer) for you and will return the data to you. If the DNA you need sequenced is too large or too complicated for that simple service, then you will need to perform some steps to prepare simpler samples (e.g. PCR amplify small target regions, or shotgun-clone your sample) before you submit them here.

The sequencer typically gives 800+ nt (or more) read lengths FOR APPROPRIATELY PURIFIED AND CONCENTRATED DNA SAMPLES. We will repeat any samples that fail due to problems in our own procedures or instruments, but we do not accept responsibility for poor results arising from improperly prepared templates or primers, or from improperly designed experiments.  See the rerun policy for further information.

The comment about APPROPRIATELY PURIFIED AND CONCENTRATED DNA samples is an important one. The quantity and quality of your DNA prep will almost always be the limiting factors in your read lengths. Template concentration is very important and it's easy to make a mistake. Contaminants are an issue too. For example, contaminants that are known to cause serious problems include:

  • Multiple templates

  • free NTPs

  • EDTA

  • Divalent cations

  • Detergents

  • Primers from previous steps (e.g. PCR primers)

Fragment Analysis

Quality Assurance: Procedures used to minimize the likelihood of errors

  • The customer enters the sample-specific information digitally at submission time so that sample names are not retyped.

  • The customer prepares fragment analysis samples for loading onto the sequencer and is responsible for optimizing reactions for the 3130xl and for the samples being analyzed.  NOTE: Even if you have gotten acceptable results from an AB 3730, you must optimize for the 3130xl.  They are different instruments.

  • We are available to advise customers on the best way to prep samples for the best results and frequently run test samples for optimization at no charge.

  • The instruments are well maintained and microsatellite installation standards are run periodically and on the rare occasion when we suspect a problem with the instrument or array.

Quality Control: Measures implemented to detect problems after the fact

  • If traces are suspect, for example if suspect samples occur consistently in one capillary, microsatellite installation standards are run with no change in running buffer, polymer or array following the suspect runs.

  • If there are no problems with the instrument, the results are sent to the customer with questions and/or suggestions to improve the traces.

  • If instrument problems are detected, they are corrected and the samples rerun at no charge.  The best results are sent to the customer.

 

Rerun Policies

Sequencing:

So - you've considered all the possibilities, realizing that your sample results have only been returned to you after the sequencing standard is verified, and you believe the problem is in the Core's handling of your samples.

While we are proud of our exceptionally low rate of error, we are human. If the error was ours, we'll certainly do whatever we can to correct the situation. We'd be glad to rerun the sample to find out if there was a problem, with the following conditions:

  • You must contact us as soon as possible - within 2-3 days, so that there is a good chance we still have your original samples.

  • We will need to resequence the original sample in its original tube. Sorry, but you may not substitute samples or make fresh dilutions. If that were allowed, We would not be able to determine whether we really made the mistake or not.

  • If the repeat sequencing works, then clearly we blew it the first time and you will not be charged for the rerun.

  • If however your sample fails a second time, then we must assume the problem was with your sample and we must charge you for the rerun.  As many of you know, we are quite willing to work with you on troubleshooting your reactions and frequently don’t charge you until we get your sequence.  If you’re not willing to work with us, we must assume that you know the problem is with your sample and that you are working to solve the problem yourself.

Please note: two "identical" sequencing reactions may differ slightly, but noticeably. A rerun may produce marginally better results than the first run, but if in our judgment it doesn't indicate that we goofed on the first run, we will assume we did not.

Fragment Analysis:

If we have an indication of a problem with the instrument, we will rerun the originally submitted samples at no charge when we have solved the problem.  We will send the best results we get.  If samples are resubmitted for rerun they will be charged as new samples.

 



 

To initiate a rerun, please email us stating that you have read the above and agree to the conditions.  State exactly which sample numbers you wish to have repeated.  Also please describe what you think the problem was with the initial run.

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