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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16 | JANUARY 15, 2018 | GENengnews.com | Genetic Engineering & Biotechnology News Madelyn Light Next-generation sequencing (NGS) has brought about a revolution in the detection of genetic abnormalities. Its unprecedented accuracy, combined with speed and afford- ability, has made NGS a commonly used technique, both in clinical and research en- vironments. The success of NGS in diagnostics and research has made it an attractive analysis option, not just for use with fresh tissue sam- ples, but also with older, archived specimens. Many labs have extensive archives contain- ing well-documented tissue specimens, which can be a valuable resource. A potential issue with archived specimens is that they are often processed for histology directly after retrieval. These formalin-fixed, paraffin-embedded (FFPE) samples retain their structure for long periods of time, but the process may also damage or alter the state of the DNA inside the cells. Here, we discuss how measuring DNA quality can help optimize coverage in NGS, to get the most out of your FFPE samples. Damaged by the Process The process of tissue fixation and embed- ding requires subjecting a sample to a range of processing steps that can affect DNA qual- ity. These steps include formalin fixation, dehydration (with potential for contamina- tion), and exposure to high temperatures for several hours. Furthermore, once samples are embedded, long-term storage can lead to spontaneous degradation of DNA. The most critical aspect when retrieving DNA from FFPE samples is to break the crosslinks between DNA and other mol- ecules. These crosslinks, introduced during the formalin fixation, affect DNA mol- ecules—binding them either to themselves or to proteins. Inadequate de-crosslinking means less DNA will be available for down- stream analysis. DNA extraction kits pro- vide protocols with detailed guidance on choosing the right parameters for lysis and de-crosslinking. Library Complexity The main parameter affected by the quality and quantity of DNA available for sequencing is library complexity. Adequate library complexity is essential for sufficient target coverage, to achieve sensitive detec- tion of somatic variants. PCR-based am- plification helps to increase the quantity of DNA, but not necessarily the complexity of the final library. Figure 1 shows the effects that the quality and quantity of the retrieved DNA have on the sequencing library. A range of commercially available DNA extraction kits can be used to lyse FFPE Strategies for Rescuing DNA from Preserved Samples Opening Up the FFPE Archives OMICS Assay Tutorial Figure 1. Quality and quantity of DNA affect library complexity. (A) Starting with sufficient high-quality DNA produces a representative, high-complexity library. (B) Low amounts of starting material produce a low-complexity library that is not completely representative of the starting sample. (C) A poor-quality sample produces a low-complexity library, because damaged DNA is not converted during library construction. (D) Increasing the amount of DNA can potentially compensate for low quality and improve library complexity. Figure 3. (A) The relationship between DIN and maximum mean coverage. (B) Variation of coverage depth with DNA input. (C) Minimum DNA input for 500× mean coverage. (D) Maximum mean coverage from 50 ng input DNA. Figure 2. DNA extraction for five archived FFPE blocks. Total yield was assessed using the Qubit® dsDNA BR Assay Kit (Thermo Fisher Scientific), and DIN was determined using the Agilent TapeStation® genomic DNA analysis tape screen.

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