Genetic Engineering & Biotechnology News

JAN15 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | | JANUARY 15, 2018 | 17 samples and extract the DNA. To investigate whether it is possible to improve quality of extracted DNA by choice of kit, we assessed the DNA retrieved from five FFPE samples using four different kits (Figure 2). We looked both at the amount of DNA extracted as well as the DNA integrity number (DIN), which is used as a measure of DNA quality. Our results show that both yield and DIN are unaffected by the choice of extraction kit, indicating that fixation and state of the sam- ple are the factors that most affect the quality of a DNA sample and, as a whole, the quality of the resulting sequencing library. So, how can one rescue a low-quality sample? How Quality and Quantity Affect Target Coverage After checking the integrity of the re- trieved DNA, we investigated how this translated to target coverage. We took 10 ng of DNA from each sample, prepared libraries using the KAPA Hyper Prep Kit (Kapa Biosystems), and performed target enrichment using the xGEN ® AML Can- cer Panel (Integrated DNA Technologies). The results are plotted as coverage vs. DIN (Figure 3A). Figure 3A shows that the maximum mean coverage corresponds very well with the DIN score. This means that assessing the condition of extracted DNA is a good pre- dictor of library complexity. Maximum mean coverage is an estimate of how many unique molecules will align to a target region when the library undergoes deep sequencing, and is defined as the av- erage target coverage depth that would be expected when sequencing deeply enough to observe 75% PCR duplicates. Highly complex libraries have many unique mol- ecules and can achieve very deep coverage. Low complexity libraries, which contain mostly PCR duplicates, will have very shallow unique coverage. Unique cover- age is an important factor in determining an assay's ability to detect low-frequency mutations. Is it possible to improve coverage by in- creasing the amount of input DNA? To find out, we varied the amount of input DNA be- tween 1 and 100 ng, corrected the difference with the number of preamplification PCR cycles, and plotted maximum mean coverage against input DNA. The dotted lines in Figure 3B represent linear regression of DNA with different lev- els of integrity. The lines show that there is a near-linear relationship between coverage and input amount, which means that the cov- erage of low-quality DNA can be improved by using more starting material. Figure 3C shows how much DNA we needed to get 500× mean coverage, and Figure 3D shows how much coverage we got from 50 ng of starting material. Our results indicate that if DNA quality is poor, even 100 ng might not be sufficient to get to 500× coverage. DNA Quality a Good Predictor of Library Complexity Archived FFPE samples can be a valuable resource for studying genetic abnormalities with NGS, but the condition of the extracted DNA is a limiting factor for target cover- age and therefore, data quality. Measuring the DNA quality after extraction is a good predictor of the complexity of the resulting library, and this information can be used to optimize the sequencing run. This can be done by using DIN, but other commercially available kits, such as the Q score (Kapa Bio- systems), work just as well. It is clear that fixation, embedding, and recovery have a negative impact on DNA integrity and therefore, library complexity. We have demonstrated here that measur- ing DNA quality is an important step in sequencing from FFPE samples. Samples with less-than-optimal DNA can be rescued by increasing the amount of starting mate- rial. It is even possible to estimate coverage based on the quality measurement and the chosen input amount. These results can help clinicians and researchers harness the power of their FFPE samples in their quest to advance understanding of genetic abnor- malities. Sign up for TOC Alerts The only peer-reviewed journal focused on the rapidly moving fi eld of 3D printing and related technologies and related technologies Editor-in-Chief: Skylar Tibbits Madelyn Light ( is a scientist in the NGS Scientific Applications group at Integrated DNA Technologies. Website: OMICS Assay Tutorial

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