Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Genome Sequencing from Post-Mortem Formalin-Fixed, Paraffin-Embedded Lung Tissues
2021; Elsevier BV; Volume: 23; Issue: 9 Linguagem: Inglês
10.1016/j.jmoldx.2021.05.016
ISSN1943-7811
AutoresClaude Van Campenhout, Ricardo De Mendonça, Barbara Alexiou, Sarah De Clercq, Marie-Lucie Racu, Claire Royer-Chardon, Ștefan Rusu, Marie Van Eycken, Maria Artesi, Keith Durkin, Patrick Mardulyn, Vincent Bours, Christine Decaestecker, Myriam Remmelink, Isabelle Salmon, Nicky D’Haene,
Tópico(s)COVID-19 diagnosis using AI
ResumoImplementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, 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 of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (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 optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for 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. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions. Implementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, 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 of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (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 optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for 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. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions. The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses are a family of enveloped single-strand, positive-sense RNA viruses that cause a wide spectrum of respiratory diseases. Since the initial report on this novel coronavirus in Wuhan, China,1Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. Xie X. Yin W. Li H. Liu M. Xiao Y. Gao H. Guo L. Xie J. Wang G. Jiang R. Gao Z. Jin Q. Wang J. Cao B. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395: 497-506Abstract Full Text Full Text PDF PubMed Scopus (31672) Google Scholar, 2Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. 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Molecular detection of SARS-CoV-2 infection in FFPE samples and histopathologic findings in fatal SARS-CoV-2 cases.Am J Clin Pathol. 2020; 154: 190-200Crossref PubMed Google Scholar showed the feasibility of SARS-CoV-2 sequencing on FFPE blocks, but only one case of two was contributory. The present study aimed to optimize SARS-CoV-2 genome sequencing using NGS on 22 post-mortem FFPE tissues. Lung samples were collected from the 16 first confirmed COVID-19 (positive RT-qPCR assay on nasopharyngeal swab and/or bronchoalveolar lavage) patients who died in Hôpital Erasme (Brussels, Belgium) since March 13, 2020, and with a positive SARS-CoV-2 E gene RT-qPCR on lung FFPE blocks (see below). The study protocol was approved by the local ethics committee (P2020/218). The autopsy procedure, clinical courses, and histopathologic findings have been already described.5Remmelink M. De Mendonça R. D'Haene N. De Clercq S. Verocq C. Lebrun L. Lavis P. Racu M.L. Trépant A.L. Maris C. Rorive S. Goffard J.C. Dewitte O. Peluso L. Vincent J.L. Decaestecker C. Taccone F.S. Salmon I. Unspecific post-mortem findings despite multiorgan viral spread in COVID-19 patients.Crit Care. 2020; 24: 495Crossref PubMed Scopus (199) Google Scholar Briefly, six samples per lung lobe (ie, a total of 30 samples) were collected, formalin fixed, and paraffin embedded (except for two patients who had previously undergone lobectomy for cancer and for whom only 18 samples were taken). One or two blocks were randomly selected for molecular analysis among FFPE blocks showing histopathologic lesions. When two blocks were tested, they included one FFPE block from the left lung and one FFPE block from the right lung, to evaluate the heterogeneity of viral spread. Moreover, one sample was snap frozen for each lung lobe. The material was biobanked by the Biobanque Hôpital Erasme-Université Libre de Bruxelles (BE_BERA1), Cliniques Universtaires de Bruxelles Hôpital Erasme, Biobanking and Biomolecular Ressources Research Infrastructure-European Research Infrastructure Consortium. Semiquantitative evaluation of hemorrhage on hematoxylin and eosin slides was performed by two senior pathologists (N.D. and M.R.) as follows: negative or 30% of lung parenchyma showing intra-alveolar hemorrhage (+++). Necrosis was evaluated as follows: negative (0) or positive (+). For FFPE blocks, total nucleic acids were extracted from two unstained slides (10 μm thick) using the Maxwell RSC DNA FFPE kit and the Promega Maxwell extractor following the protocol described by the manufacturer (Promega Corp., Madison, WI) in an elution volume of 50 μL. For frozen tissues, RNAs were extracted using PureLink RNA Mini Kit (ThermoFisher Scientific, Waltham, MA) following manufacturer's instructions. The RNA yield was quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific). For FFPE blocks, RNA quality was analyzed with the Agilent RNA 6000 Pico Kit on a Bioanalyzer 2100 (Agilent, Santa Clara, CA). The RNA from the FFPE blocks showed a fragmented profile, with a mean peak height of 130 nucleotides. The mean percentage of RNA fragments >200 nucleotides was of 60%, and no samples showed a percentage of RNA fragments >200 nucleotides of 99% coverage of the SARS-CoV-2 genome, covering from position 43 to position 29,842 (positions related to reference sequence3Wu F. Zhao S. Yu B. Chen Y.M. Wang W. Song Z.G. Hu Y. Tao Z.W. Tian J.H. Pei Y.Y. Yuan M.L. Zhang Y.L. Dai F.H. Liu Y. Wang Q.M. Zheng J.J. Xu L. Holmes E.C. Zhang Y.Z. A new coronavirus associated with human respiratory disease in China.Nature. 2020; 579: 265-269Crossref PubMed Scopus (7235) Google Scholar). Amplification condition was 98°C for 2 minutes for initial denaturation, followed by 20, 25, or 30 cycles (Supplemental Table S1) at 98°C for 15 seconds and 60°C for 4 minutes. Then, the amplicons were digested, barcoded, and purified using AMPure XP Beads (Beckman Coulter, Brea, CA). The libraries were amplified by PCR, and size selection was performed using AMPure XP Beads. The Ion 510, Ion 520, and Ion 530 Kit, Chef and the Ion Chef (ThermoFisher Scientific), were used for template preparation and chip loading. Sequencing was performed using the S5 Gene Studio instrument (ThermoFisher Scientific). SARS-CoV-2 whole-genome was sequenced using Oxford Nanopore (Oxford, UK) technology as previously described.19Artesi M. Bontems S. Göbbels P. Franckh M. Maes P. Boreux R. Meex C. Melin P. Hayette M.P. Bours V. Durkin K. A recurrent mutation at position 26340 of SARS-CoV-2 is associated with failure of the E gene quantitative reverse transcription-PCR utilized in a commercial dual-target diagnostic assay.J Clin Microbiol. 2020; 58: e01598-20Crossref PubMed Scopus (127) Google Scholar The raw sequencing data were analyzed using the torrent suite software version 5.12 (ThermoFisher Scientific). The sequencing metric analysis was performed using the coverage analysis plug-in. For fresh samples, the manufacturer (ThermoFisher Scientific) recommends obtaining 1 M reads per sample and reports that the uniformity is >85%. The following sequencing quality classification was used: optimal if the mapped reads were >1,000,000 and uniformity >90%; suboptimal if the mapped reads were between 1,000,000 and 500,000 and/or uniformity between 80% and 90%. If the mapped reads were <500,000 and/or uniformity between 55% and 80%, the sequencing quality was considered as poor. If the uniformity was 90% and recurrent variants (Supplemental Table S2) reported in the literature21Pachetti M. Marini B. Benedetti F. Giudici F. Mauro E. Storici P. Masciovecchio C. Angeletti S. Ciccozzi M. Gallo R.C. Zella D. Ippodrino R. Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant: version 2.J Transl Med. 2020; 18: 179Crossref PubMed Scopus (615) Google Scholar, 22Yin C. Genotyping coronavirus SARS-CoV-2: methods and implications.Genomics. 2020; 112: 3588-3596Crossref PubMed Scopus (189) Google Scholar, 23Stefanelli P. Faggioni G. Lo Presti A. Fiore S. Marchi A. Benedetti E. Fabiani C. Anselmo A. Ciammaruconi A. Fortunato A. De Santis R. Fillo S. Capobianchi M.R. Gismondo M.R. Ciervo A. Rezza G. Castrucci M.R. Lista F. on Behalf of Iss Covid-Study GroupWhole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe.Euro Surveill. 2020; 25: 2000305Crossref Scopus (109) Google Scholar, 24Wang C. Liu Z. Chen Z. Huang X. Xu M. He T. Zhang Z. The establishment of reference sequence for SARS-CoV-2 and variation analysis.J Med Virol. 2020; 92: 667-674Crossref PubMed Scopus (322) Google Scholar were verified in the Integrative Genome Viewer (IGV) from the Broad Institute (http://www.broadinstitute.org/igv, last accessed November 9, 2020).25Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative genomics viewer.Nat Biotechnol. 2011; 29: 24-26Crossref PubMed Scopus (8539) Google Scholar Sequences were aligned using the MUSCLE algorithm.26Edgar R.C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity.BMC Bioinformatics. 2004; 5: 113Crossref PubMed Scopus (6385) Google Scholar Clades were allowed according to GISAID definitions (ie, clade G for patients with C241T, C3037T, and A23403G variants; clade GR for patients with C241T, C3037T, A23403G, and GGG28881AAAC variants; and clade GH for patients with C241T, C3037T, A23403G, and G25563T variants). The occurrence of variants was checked on the GISAID (using CoVsurver) and Nextstrain websites to detect new variants. Viral sequences from eight patients with 90%) was also analyzed for each block and considered as independent. The U-test was applied for the comparison of two independent groups of ranked data. The Friedman test was applied for the comparison of multiple dependent groups. Spearman correlation analysis was used to analyze the relationship between the RT-qPCR Cp values and uniformities. Statistical analyses were performed using Statistica 7.1 (Statsoft, Tulsa, OK). This study included 16 confirmed COVID-19 deceased patients with a positive SARS-CoV-2 E gene RT-qPCR on lung FFPE blocks. For six patients, two different lung lobes were tested, leading to 22 FFPE blocks. RT-qPCR Cp values for the different FFPE blocks ranged from 16.02 to 34.16 (Supplemental Table S1). For SARS-CoV-2 genome sequencing, the Ion AmpliSeq SARS-CoV-2 Research Panel was used, which is an amplicon-based library preparation method. Because the 22 FFPE blocks were relatively heterogeneous in terms of RT-qPCR Cp values, three different numbers of target amplification cycles were tested: 20, 25, and 30 PCR cycles for all the blocks. Libraries suitable for sequencing were obtained for all blocks, except for one (block 2-2) for which the library concentration at 20 PCR cycles was too low for sequencing (Supplemental Table S1). Globally, no significant differences were observed in terms of sequencing metrics (number of mapped reads and coverage) with increased numbers of PCR cycles. Only the uniformity appears higher at 20 PCR cycles (median, 95.72%) than at 25 and 30 PCR cycles (medians, 92% and 88%, respectively; Friedman test: P = 0.04) (Supplemental Table S1). Next, the analyses were refined according to the RT-qPCR Cp values (Figure 1). For blocks with low RT-qPCR Cp values ( 30, a similar but slighter variation appeared in the number of mapped reads but was not significant (Friedman test: P = 0.135). Uniformity clearly decreased with the increase of the RT-qPCR Cp value for the three tested conditions (20, 25, and 30 cycles), as confirmed by the negative Spearman correlations (Figure 2). In particular, for the seven blocks with an RT-qPCR Cp value >30, five showed a uniformity of <55% for all the tested conditions. These five blocks were considered as non-contributory; 17 blocks were thus considered as contributory. These 17 contributory blocks were coming from 13 patients (including four patients with two blocks tested).Figure 2Dot plot of SARS-CoV-2 genome uniformity against RT-qPCR crossing point (Cp) values. For the same block, three different library preparation protocols were tested by varying the number of target amplification PCR cycles. The different conditions are indicated as follows: circle, 20 PCR cycles; square, 25 PCR cycles; diamond, 30 PCR cycles. Spearman correlation between RT-qPCR Cp value and uniformity is −0.63 (P = 0.002) at 20 cycles, −0.86 (P < 10−6) at 25 cycles, and −0.81 (P = 0.000006) at 30 cycles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the 17 contributory blocks, the aim was to establish the best PCR condition for sequencing performance and variant analyses. As sequencing quality criteria, the uniformity was selected as the most important factor because it is related to the homogeneity of the coverage distribution. The PCR condition with the highest uniformity was thus selected. If there were conditions with similar uniformities (±3%), the condition with the highest number of mapped reads was selected. If there were conditions with similar uniformities (±3%) and number of mapped reads (±20%), the condition with the fewest PCR cycles was preferred (Supplemental Table S1). This allowed selection of 20 PCR cycles for blocks with an RT-qPCR Cp value 30, with most of them being non-contributory (Supplemental Table S1). After adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values for the 17 contributory blocks, the median number of mapped reads and uniformity were 1,642,150 (minimum-maximum: 305,249 to 2,094,563) and 95.9% (minimum-maximum: 81% to 98%), respectively. The sequencing quality was considered as optimal for 10 blocks (Materials and Methods), with a median number of mapped reads of 1,748,009, a median coverage of 10,644, and a median uniformity of 96.4% (Table 1). The RT-qPCR Cp value of these 10 FFPE blocks varied from 18.69 to 31.14. The sequencing quality was considered as suboptimal for six blocks, with a median number of mapped reads of 1,128,420, a median coverage of 5385, and a median uniformity of 89%. The RT-qPCR Cp values ranged from 16.02 to 30.55. The sequencing quality of one block was considered as poor (Table 1). According to the RT-qPCR Cp values, significant differences were observed between contributory blocks with an RT-qPCR Cp value <24 and those with an RT-qPCR Cp value between 24 and 30 in
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