SARS-CoV-2 genome sequencing from post-mortem formalin-fixed, paraffin-embedded lung tissues

Van Campenhout, Claude; De Mendonça, Ricardo; Alexiou, Barbara; De Clercq, Sarah; Racu, Marie-Lucie; Royer-Chardon, Claire; Rusu, Stefan; Van Eycken, Marie; Artesi, Maria; Durkin, Keith; Mardulyn, Patrick; Bours, Vincent; Decaestecker, Christine; Remmelink, Myriam; Salmon, Isabelle; D’Haene, Nicky

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 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 (i.e. with a uniformity >55%) for 17 FFPE blocks. 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; i.e. 20 PCR cycles for blocks with a Cp value <28 and 25 PCR cycles for blocks with a Cp value between 28 and 30. The majority of blocks with a Cp value >30 were non-contributory. Comparison of matched frozen and FFPE tissues revealed discordance for only three FFPE blocks, all with a 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.