Recommendations to mitigate FFPE-associated problems in next-generation sequencing of FFPE-extracted DNA

Study ID Alternative Stable ID Type
EGAS00001005757 Other

Study Description

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most widely available material for diagnostics and retrospective clinical studies. Reliable sequencing data obtained from such specimens would have enormous potential for basic and clinical research. However, formaldehyde fixation introduces chemical modifications which ultimately can lead to false interpretations. Here we describe a start-to-end approach to mitigate FFPE-problems comprising preanalytical sample specification and quality, repair treatments, analytical sample preparation, and bioinformatic sub-processes. We have also developed an alternative sequential DNA repair technique (IQBErepair) addressing the cause of formalin-induced false positives: too low and uneven coverage. For low-input approaches this technique increased the unique on-target bases by 53% and 80% in two independent centers, respectively. Furthermore, we present other measures to reduce FFPE artifacts: replicate strategies (up to 98% for 1% VAF detection threshold), bioinformatic filtering (>99.9%). Combined with general recommendations for ... (Show More)

Study Datasets 1 dataset.

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Dataset ID Description Technology Samples
42 NGS libraries of a 13y/o FFPE sample, a tissue-and-patient-matched FF sample, and a GIAB sample (NA12878). In technical replicates (untreated DNA, treated DNA, two different library types, at least library duplicates for each case). Illumina NextSeq, HiSeq and NovaSeq paired-end sequencing.
Illumina HiSeq 4000,Illumina NovaSeq 6000,NextSeq 500 42

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