SARS-CoV-2 sequencing from FFPE tissues
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.
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.