Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 15 Additional quality control was done after amplification of the ligated libraries and after amplification of the capture libraries. All three steps were analyzed with the QIAxcel DNA Screening Kit using method AM320. The analysis protocol parameters were: baseline filter 160 sec, minimum distance 5 sec, and threshold 10 S/N. The run, reference marker table (RMT), and analysis parameters were defined as a standard protocol that was saved with ScreenGel® software and used for all of the runs. The capture hybridization was carried out at 65°C for 48 h. Libraries were quantified for pooling using qPCR. Sequencing was performed on an Illumina MiSeq using 150 bp paired-end reads and multiplexed into two runs of 10 samples each. Putative mutations were validated with Sanger sequencing in a CPA-accredited laboratory using an Applied Biosystems® 3500xL Genetic Analyzer. Results Per the SureSelect Target Enrichment protocol for Illumina Multiplex sequencing, quality control was performed after shearing of the genomic DNA (Figure 1), amplification of the adapter-ligated libraries (Figure 2), and after amplification of the captured libraries (Figure 3). The QIAxcel Advanced System allows complete analysis of 12 samples, including reporting, in 8 min. The results are presented as a gel-like image, electropherogram, and result table with different requested parameters (in this case, size and concentration of libraries). A B Figure 1. Analysis of sheared genomic DNA. A Lanes 1–8: Genomic DNA sheared using a Covaris e220 sonication platform was analyzed using the QIAxcel DNA Screening Kit and method AM320. QX Alignment Marker 15 bp/5 kb (marked in green) was run simultaneously with all of the samples. Lane 9: The QX DNA Size Marker 100 bp – 2.5 kb was used for precise size and concentration assessments. The image shows a smear in the range of 50 – 350 bp. B The electropherogram shows the distribution. The medium peak size is 200 bp. 1 2 3 4 5 6 7 8 9 1 5 2 0 0 4 0 0 6 0 0 1 5 0 0 5 0 0 0 15 bp 5000 bp 201 bp Size (bp) 123456789

Pages Overview