SARS-CoV-2 genome sequencing from post-mortem FFPE lung tissues

Implementation of SARS-CoV-2 testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed and paraffin-embedded (FFPE) samples. So far, one study reported the feasibility of SARS-CoV-2 genome sequencing on FFPE tissues with only one contributory case out of two. The present study aimed to optimize SARS-CoV-2 genome sequencing using the Ion AmpliSeq™ SARS-CoV-2 Panel (ThermoFisher) on 22 FFPE lung tissues from 16 deceased COVID-19 patients. SARS-CoV-2 was detected in all FFPE blocks using a real-time RT-qPCR targeting the E gene with Crossing Point (Cp) values ranging from 16.02 to 34.16. Sequencing was considered as contributory for 17 FFPE blocks, all non-contributory blocks (due to uniformity <55%) being characterized by an RT-qPCR Cp value >30. Adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values allowed to optimize the sequencing quality for the contributory blocks. Sequencing quality is also influenced by the presence of hemorrhage and/or necrosis in the tissues. Comparison of matched frozen and FFPE tissues revealed discordance only for three FFPE blocks, all with a suboptimal sequencing result and with a RT-qPCR Cp value >28. Variant identification and clade classification was possible for 13 patients. The present study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathological lesions.

病理实验室日常开展严重急性呼吸综合征冠状病毒2（SARS-CoV-2）检测时，需对检测流程进行适配，以处理福尔马林固定石蜡包埋（Formalin-Fixed and Paraffin-Embedded, FFPE）样本。迄今为止，仅有一项研究报道了在FFPE组织上开展SARS-CoV-2基因组测序的可行性，其2例样本中仅1例可获得有效测序数据。本研究旨在针对16名已故COVID-19患者的22份FFPE肺组织样本，采用Ion AmpliSeq™ SARS-CoV-2测序面板（赛默飞世尔科技，ThermoFisher）优化SARS-CoV-2基因组测序流程。本研究通过靶向E基因的实时逆转录定量聚合酶链反应（real-time RT-qPCR）对所有FFPE组织块进行检测，其交叉点（Crossing Point, Cp）值介于16.02至34.16之间。17份FFPE组织块可获得有效测序数据，所有无法获得有效数据的组织块（其测序覆盖均匀度<55%）的RT-qPCR Cp值均大于30。根据RT-qPCR Cp值调整靶标扩增PCR循环数，可优化有效测序样本的测序质量。组织中存在出血和/或坏死也会对测序质量产生影响。对配对的冰冻组织与FFPE组织进行比对后发现，仅3份FFPE组织块的检测结果存在不一致，这3份样本的测序结果均欠佳，且RT-qPCR Cp值均大于28。13名患者可实现病毒变异株鉴定及进化分支（clade）分类。本研究验证了FFPE组织块上SARS-CoV-2基因组测序的可行性，并为探索病毒基因型与组织病理学病变之间的关联提供了可能。