Quality Assurance/Quality Control in the U of M DNA Sequencing Core
We take great pride in our low error rate, and we 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 Core-induced mistakes.
These measures are detailed here.
Quality Assurance: Procedures used to minimize the likelihood of errors.
- All samples are checked by a Sample Clerk for accurate labeling and similar
client errors before we accept them for processing. That Clerk maintains
custody of the samples in our collection until they are turned over to the
technicians for processing.
- Computerized sample tracking ensures reliable flow control. Only the client
can enter the sample-specific information at submission time. Data generated
at the completion of a sequencing run is automatically and reliably associated
with the correct sample tracking number, and returned to the correct client.
- Pipetting is performed with a worksheet detailing which sample goes with
which primer. Technicians use handwritten coding systems to organize their
samples for each set, and to minimize the likelihood of mis-pipetting.
- All templates are added to an upper corner of the well and visually verified
before they are spun to the bottom.
- All primers are similarly added to the top, for visual verification.
- Premixes are prepared with all reaction components except template and primer
so that the exact same cocktail is present in all wells (including standards).
- The final volume of the well is visually checked to verify the presence of
- The volume of the plate is scanned visually after the purification step (Seph
columns or CleanSEQ) to verify consistency of volume, before they are loaded
onto the sequencer.
- After the run, all results are scanned one-by-one to check for symptoms of
problems or of common failures (premix error, sequencer malfunction, salt effect,
- All plates are pipetted in a rotationally-asymmetric pattern, and differing
from plate to plate, to allow for reliable detection of plate swapping or
plate rotation errors.
- If a standard fails or generates inappropriate sequence, then an automatic
warning is generated, facilitating detection of plate rotation, swapping
errors, mis-pipetting, premix errors, etc.
Quality Control: Measures implemented to detect problems after the fact.
- Each set of samples includes at least one standard with known purity and known
performance (and often several). If any standards do not sequence properly, it
suggests a possible problem.
- All samples are passed through a scanning system that scores them for read length
and quality of basecalls. We can detect unusual decreases in our read lengths or
our quality scores. We can also produce reports detailing the performance with
respect to technician, sequencer, client, time/date interval, etc.
- All sample sets are scanned automatically to detect the presence of suspicious
similarities between samples from adjacent capillaries. This detects any instance
of capillary crosstalk.
- All results are run though an 'auto-comment' system, that can detect certain
modes of failure. Although it is not reliable enough for generating the client
feedback, this can be useful for grasping overall views of failure modes.
- Samples that fail under suspicious circumstances (singlet failures among larger
sets of otherwise-successful samples; large-scale failures from otherwise-savvy
clients; 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.
- Clients are offered a way to request repeats when they can't come up with an
explanation for a failure. This is the last-resort detection of a problem in
our processing. Client-requested repeats that identify improperly processed
samples are the most direct metric of our errors.
- Core administrators frequently view global reports of read lengths, failure
patterns, client success rates, etc. These sometimes suggest additional Q/C
checks to be performed. Frequently these lead to discussion with the client to
encourage additional diagnosis of any perceived problems (whether client- or
- Occasional large-scale reassessments are conducted, during which the Director
scans through a large block of samples to detect instances where the technicians'
performances might be called into question.