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. 2013 Jun 6;8(6):e62692. doi: 10.1371/journal.pone.0062692

Figure 5. DNA qualification for next-generation sequencing applications.

Figure 5

Effect of low-quality DNA on next-generation sequencing (NGS) workflow. Three FFPE samples were tested for construction of NGS amplicon libraries (Ion Torrent Ampliseq Cancer Panel). Qubit: 40 ng of DNA according to Qubit measurement were processed using the Ampliseq library construction kit (multiplex PCR amplification of 191 DNA regions from 46 cancer-related genes). NanoDrop: absorption spectra of samples showed different degrees of organic contamination (230 nm spike, A260/A230 ratio). Agilent: quality and quantity of the obtained libraries were evaluated by Agilent high sensitivity assay on-chip electrophoresis, where the library is represented by the large band between 150 and 200 bp. Fragments test: histogram showing length and abundance of produced sequences. Sample FFPE 5 did not produce a good library due to high organic contamination; this is revealed by the remarkable spike at 230 nm that concurs to the low 260/230 ratio, and explains the faint electrophoretic band and the almost flat fragments test histogram.