Fig. 5

DNA fragmentation in FFPE bacteria. a Evaluation of DNA integrity with fragment analyser. Electropherograms of DNA purified from Protoblocks with a mix of 5 bacterial strains (red) and Protoblocks loaded with Escherichia only (yellow) and compared with matched NF bacterial mix (blue) and Escherichia (green). NF bacterial DNA had a higher integrity (GQN > 6.6), while FFPE bacterial DNA from either sample was highly fragmented (GQN ≤ 0.1). No significant difference was observed between Protoblocks or NF samples. GQN = % of DNA above the threshold. The GQN threshold (dotted line) was set to that used for sequencing libraries (10,000). b Measuring the recovery of PCR readable DNA from FFPE bacteria in Protoblocks by qPCR. (i) Schematic of primer design for targeted fragments. Both 200 bp and 460 bp DNA fragments target the same E. coli K-12 regions. (ii) PCR recovery. Box plot of DNA recovery from 460 bp (green) and 200 bp (orange) FFPE DNA fragments (for each box, n = 9) compared with NF DNA (cyan; for each box n = 6) normalised to 10⁷, 10⁵ and 10³ genomes. Mean recovery of DNA from Protoblocks compared with input DNA significantly differed in both FFPE sample types (p < 0.001) as per one-sample Wilcoxon signed-rank test. Fragment length also significantly influenced DNA recovery of FFPE samples (p < 0.001), as per Wilcoxon signed-rank test. (iii) Gram-stained slides used for confirming bacterial content. (In all cases, p = + > 0.1, * < 0.05, ** < 0.01 and *** < 0.001)