Application Guide - QIAxcel Advanced

76 QIAxcel Advanced Application Guide 10/2016 Results and Discussion MIRU-VNTR loci The 15-locus panel for epidemiological studies and the 24-locus panel for phylogenetic resolution were used as the standard sets for identification of the main mycobacterial genotypes (Table 1). Other locus panels can be used when addressing specific lineages, such as Beijing strains. VNTR allele-calling accuracy The overall concordance on allele calling observed between the two methods is 1803/1824 (98.8%). For the size range of 140–900 bp, it is 99.9%. We observed 21 discrepancies: 19 on locus VNTR4052 (alleles 8 to 10), 1 on locus VNTR3690 (allele 8), and 1 on locus VNTR1955 (allele 15). Sanger sequencing confirmed all the allele sizes obtained by conventional gel electrophoresis. The QIAxcel data showed overestimated sizes for alleles above 900 bp. Optimization of the sizing algorithm for OM1700 is in progress. An in-house allelic ladder can act as an alternative internal size marker for accurate allele calling. Influence of DNA concentration on sizing It has been proved that conventional gel electrophoresis is negatively affected by high DNA concentrations. Therefore, we investigated the influence of DNA concentration on the resolution and size estimation for the two methods, and compared the results (Figure 1). Dilution series of the same PCR products were used to estimate the impact of DNA concentration on sizing accuracy. The results show no difference in allele calling at concentrations between 0.4 and 106 ng/μl. The QIAxcel Advanced System enables detection of DNA at concentrations as low as 0.4 ng/μl. The limit of detection using the conventional method is 2.2 ng/μl. Thus, PCR DNA quantification and/or sample dilution are not required before the QIAxcel run under these conditions. Reproducibility and repeatability A number of variables (operator, different alignment marker and cartridge batches, and cartridges of different ages) were tested using a ladder of previously sequenced amplicons of known sizes in order to cover the broad size range (100, 206, 382, 348, 562, 639, 681, 1065 bp). Tests were done with two cartridges. The maximum deviation for the estimated sizes ranged between 1.9 bp (100-bp amplicon) and 6.5 bp (1065-bp amplicon). The results indicate that the automated analysis with the QIAxcel protocol is repeatable (0.4 and 0.8% at the beginning and end of kit shelf life, respectively) and reproducible (0.4 and 1.2%) for MIRU- VNTR genotyping. A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 – 3000 – 1000 – 700 – 500 – 300 – 100 2000 – 800 – 600 – 400 – 200 – 15 – [bp] [bp] QIAxcel OM1700 Conventional method Corresponding agarose-3%-gel Peak size Sample 1 Expected size: 643 bp Conc. (ng/µl) QX sizing Deviation 76 634 1.4% 38 635 1.2% 22 633 1.6% 11 637 0.9% 4.6 640 0.5% 2.2 642 0.2% 1.0 643 0.0% 0.4 645 0.3% 106 627 2.5% 35 627 2.5% 28 633 1.6% Sample 2 Figure 1. QIAxcel Advanced performs accurate DNA fragment sizing. Gel electrophoresis and fragment sizing of DNA fragments at various concentrations were performed using conventional gel electrophoresis and the QIAxcel Advanced instrument. Locus MIRU10 has repeats of 55 bp. The 643-bp fragment represents 3 alleles.

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