Fig 1.
DNA extracted from three patient groups was sequenced after treatment with and without UNG. The fixation group consisted of three patients from 2015 where paired tissue samples were snap frozen or put into formalin for a determined length of times. A baseline group consisting of 20 patients all from 2015/16 were fixed in formalin for an unknown amount of time. The block age group consisted of three patients from 1994, three from 2004 and three from 2014; all samples had an unknown fixation time. DNA was extracted from normal (N) and tumor (T) tissue when available.
Table 1.
Fixation group patient metadata.
Table 2.
Targeted regions of amplification for the NGS assay.
Fig 2.
Total reads mapped in the baseline group does not change by DNA extraction type.
GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
Fig 3.
Read quality in the baseline group does not change by DNA extraction type.
Read quality as measured by percent reads mapped. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
Fig 4.
Number of deamination variants ranges widely amongst samples when fixation time is uncontrolled.
Orange crosses represent number of deamination variants (y-axis; C->T/G->A). Blue dots represent all other possible variants. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
Table 3.
Somatic mutation profile.
Fig 5.
Sequencing read quality decreases over fixation time.
Read quality as a measure of percent reads mapped (y-axis) decreases with a longer tissue fixation time (x-axis). Blue dots represent DNA without UNG treatment (extracted using QiaAmp FFPE kit; QA), orange crossed represent matched DNA treated with UNG (extraction using the GeneRead kit; GR UNG). Linear regression line depicted by the central broken line and encompassed by dotted lines representing the 95% confidence interval (CI region colored). Frozen samples used for ground truth are represented by green dots. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
Fig 6.
Deamination mutations significantly increase at 48 hours fixation treatment time.
Analysis of deamination events in the fixation group for all time points comparing UNG (uracil-N-glycosylase) and non-UNG effects. All variants representative of possible deamination events (C->T, G->A) are denoted by the orange crosses; linear regression line shown by the broken line and encompassed by dotted lines representing the 95% CI (CI regions colored). Similarly all other variants are represented by blue dots, shading and lines. After UNG treatment of the DNA, the deamination events nearly disappear (bottom row).
Fig 7.
Deamination events increase with sample age when not treated with UNG.
GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.
Fig 8.
Read quality decreases with sample age in the age group.
GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.