DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer’s protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ² = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100–400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

从福尔马林固定，石蜡包埋的组织切片中提取的DNA通常由于产量或降解能力差而无法进行测序。我们通过使用54个生物样本按照制造商的规程测试增加的酶量和增加的消化长度来优化蛋白酶K消化，并在离心管中进行消化。蛋白酶K的量加倍导致产量中位数增加96％。将优化的蛋白酶K方案应用于显微镜载玻片上脱石蜡的切片，产量进一步提高了41％，但仅在> 50,000上皮肿瘤细胞/切片上。DNA产量现在（χ相关²= 0.84），并且可以通过上皮肿瘤细胞数量进行预测。使用终点多重PCR（在凝胶上观察到100-400 bp的扩增子），定量PCR（qPCR； Illumina FFPE QC分析）和纳米电泳（DNA完整性编号[DIN]）测定DNA完整性。通常，产量的增加伴随着完整性的提高，但有时qPCR和DIN结果是矛盾的。几乎普遍获得了400 bp的扩增子。优化过程使我们能够将未能通过公布的用于确定对全基因组测序的质量控制阈值的样品的百分比从33％降低到7％。