Greater confidence in calling low-frequency variants – from as little as 10ng of severely formalin-compromised DNA
With advances in next-generation sequencing (NGS) technology, genetic information can be extracted from an increasingly diverse range of samples. Formalin-fixed, paraffin-embedded (FFPE) storage is a standard method for archiving tissue biopsies and these biopsies can be used in NGS, for example to study cancer development and progression.
However, the quality of DNA in FFPE samples is often severely damaged and compromised compared to other methods of sample storage. Consequently, it may be difficult to distinguish between true low frequency mutations and damage-induced low frequency false positives. In these samples, hybridisation-based target enrichment (where input DNA has been sheared into short, fragments and selectively captured) provides superior performance compared to amplicon-based enrichment. This is due to the superior tolerance of the hybridisation approach for the fragmented DNA routinely found in FFPE samples. Hybridisation-based enrichment also provides greater uniformity of coverage, fewer false positives and superior variant detection due to use of fewer PCR cycles1.
To improve the sequencing results of FFPE samples even further, a DNA pre-treatment step can be introduced to address different types of DNA damage. OGT’s SureSeq™ FFPE DNA Repair Mix uses a mixture of enzymes to repair a range of DNA defects including deamination of cytosine, nicks and gaps, oxidised bases, and blocked 3’ ends.
In this study, carried out in collaboration with Horizon Discovery, formalin-compromised DNA (fcDNA) samples of differing severity were repaired with the SureSeq FFPE repair mix and sequenced using a SureSeq custom hybridisation-based panel. The aim of the study was to investigate the effect of the repair mix and also the performance of the hybridisation-based enrichment method on DNA with varying levels of damage by measuring critical parameters at different points in the sequencing protocol:
- DNA length distribution after extraction
- DNA yield immediately before capture
- Mean target coverage in sequencing
- The ability to accurately detect known variants with excellent uniformity
When studying FFPE tumour biopsies, low DNA starting material is also a frequently encountered issue. In order to study the effect of this, tests were carried out with varying amounts of DNA starting material to determine sequencing sensitivity when DNA availability is limited.
- Selecting the best NGS enrichment assay for your needs, available at: https://www.ogt.com/resources/literature/1357_selecting_the_best_ngs_enrichment_assay_for_your_needs
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