Application Guide - QIAxcel Advanced

72 QIAxcel Advanced Application Guide 10/2016 A B The increased sensitivity of the QIAxcel compared to agarose gels revealed erroneous calls in agarose gels arising from weak amplicons that could not be easily detected (Figure  2). The weak amplicons detected by QIAxcel in the original samples could only be detected by conventional gel electrophoresis after the original sample was concentrated five-fold. Although this resulted in enhanced detection of weak bands, it also led to problems visualizing other samples due to overloading. In contrast, we had no problems visualizing either the original samples or the concentrated samples with the QIAxcel instrument. The sizing accuracy and resolution of the QIAxcel compared to agarose gels revealed erroneous calls in agarose gels arising from band sizing errors (Figures 3 and 4). Slight gel anomalies may lead to erroneous calls when amplicons are sufficiently similar in size unless extreme precaution is taken to maintain repro- ducibility and quality control during electro- phoresis and during subsequent analysis of the gel image data. This is a major challenge for high-throughput environments and represents the source of all of the band size-related errors observed in our dataset (Figure 5). Figure 4. Misclassification of error calls due to poor resolution coupled with gel anomaly when analyzing samples by gel electrophoresis. Samples were analyzed either on A. the QIAxcel or B. and C. by agarose gel electrophoresis. The arrows in B. represent the bands that were misclassified as the 307 bp band due to a gel artifact. These samples were reanalyzed by agarose gel electrophoresis in C. Figure 2. False negative error calls due to weak amplicons when analyzing samples by agarose gel electrophoresis. Samples were analyzed on either A. the QIAxcel at the original concentration or B. by agarose gel electrophoresis with five-fold concentration. Samples 3 C. and 5 D. were analyzed by agarose gel electrophoresis at the original concentration (O), with five-fold concentration (C), and at the original concentration on the QIAxcel (Q). The arrows represent bands originally scored as negative. A 1 2 3 4 5 6 7 8 9 10 11 B 1 2 3 4 5 6 7 8 9 10 11 Figure 3. False positive error calls due to poor resolution when analyzing samples by agarose gel electrophoresis. Samples were analyzed either on A. the QIAxcel or B. by agarose gel electrophoresis. The arrows represent the bands of approximately 440 bp that was originally scored as positive due to its proximity to the band of expected size (486 bp). 307 bp – 282 bp – O C Q O C Q A B 486 bp – 440 bp – 1234567891011 1234567891011

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