For histological staining or archival purposes, tumor biopsies often are formalin-fixed-paraffin-embedded. Although formalin treatment preserves the tissue, it also damages nucleic acid and makes it difficult to identify true variants in tumor samples with next-generation sequencing (NGS).
The latest research used a standard pathology protocol that involved preserving tissue with 10% neutral buffered formin for 24 hours. However, there are so many FFPE tissue applications available that help people in a better understanding of FFPE tissues.
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Many clinical research studies could be dependent on archival tissue that was fixed with inconsistent methods or inconsistencies. Personal scientists based their initial handling of FFPE samples suggested that variations in sequencing results could be caused by deviations from the standard fixation procedure. This could be due to inaccuracy in logging the fixation protocol, variations in fixation time, fixation temperatures, and/or different storage conditions for the samples.
The quality of DNA extracted from cells treated with formalin varied greatly between protocols. One day of formalin fixation in 100% phosphate-buffered (PBS)-buffered formalin at ambient temperature produced DNA with high molecular mass and low fragmentation. This preparation also produced a library of high quality.
However, unbuffered formalin fixation at higher temperatures and for longer periods of time led to highly fragmented and low molecular-weight DNA. This poor quality DNA resulted also in low library efficiency.
These results show that buffered formalin is essential for preserving high-quality DNA for downstream NGS protocol. This can lead to decreased alignment metrics, as well as reduced sensitivity and specificity for somatic variant calling.