32e828639542f176f38f68e58d6b83e4 pmc_34771584.pdf 351aad977e291dd7de392c51ea20d1ca351d0fe0 pmc_34771584.pdf b550700632aebf9e269a6867daef1f89a38cd379ec9f20aa0d36f8717a2fe84b pmc_34771584.pdf Title: Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues Subject: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses. Keywords: microbiome; colorectal cancer; 16S gene sequencing; RNA in situ hybridization; FFPE Author: Alessandra Borgognone, Garazi Serna, Marc Noguera-Julian, Lidia Alonso, Mariona Parera, Francesc Català-Moll, Lidia Sanchez, Roberta Fasani, Roger Paredes and Paolo Nuciforo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Oct 29 07:51:53 2021 CEST ModDate: Fri Oct 29 08:03:04 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3130955 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WNWTFR+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 TTCDRT+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 EHOAYY+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 UZNGFI+URWPalladioL-Roma Type 1 Custom yes yes 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DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Sample Collection Destination: subsection.2.1 Item: Title: DNA Extraction Destination: subsection.2.2 Item: Title: Library Preparation for Illumina MiSeq Sequencing Destination: subsection.2.3 Item: Title: 16S rRNA Sequence Analysis Destination: subsection.2.4 Item: Title: Statistical Analysis Destination: subsection.2.5 Item: Title: Species-Level Taxonomy Inference and Phylogenetic Tree Reconstruction Destination: subsection.2.6 Item: Title: Microbial In Situ Hybridization (ISH) and Image Analysis Destination: subsection.2.7 Item: Title: Results Destination: section.3 Children: Item: Title: Data Processing and Quality Control Destination: subsection.3.1 Item: Title: Comparison of the Microbial Diversity between FF and FFPE Tissues Destination: subsection.3.2 Item: Title: Taxonomic Profiling and Discriminant Taxa between FF and FFPE Tissue Samples Destination: subsection.3.3 Item: Title: Putative Fusobacterium, Bacteroides and Propionibacterium Species Classification Destination: subsection.3.4 Item: Title: Comparison of CRC-Associated Bacteria Characterized by RNA-ISH and 16S rRNA Sequencing Destination: subsection.3.5 Item: Title: Quality Assessment of FFPE DNA Based on 16S rRNA Amplicon Profiling Destination: subsection.3.6 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Performance of 16S Metagenomic Profiling in Formalin-Fixed Paraffin-Embedded versus Fresh-Frozen Colorectal Cancer Tissues Author: Alessandra Borgognone, Garazi Serna, Marc Noguera-Julian, Lidia Alonso, Mariona Parera, Francesc Català-Moll, Lidia Sanchez, Roberta Fasani, Roger Paredes and Paolo Nuciforo Subject: Formalin-fixed, paraffin-embedded (FFPE) tissues represent the most widely available clinical material to study colorectal cancer (CRC). However, the accuracy and clinical validity of FFPE microbiome profiling in CRC is uncertain. Here, we compared the microbial composition of 10 paired fresh-frozen (FF) and FFPE CRC tissues using 16S rRNA sequencing and RNA-ISH. Both sample types showed different microbial diversity and composition. FF samples were enriched in archaea and representative CRC-associated bacteria, such as Firmicutes, Bacteroidetes and Fusobacteria. Conversely, FFPE samples were mainly enriched in typical contaminants, such as Sphingomonadales and Rhodobacterales. RNA-ISH in FFPE tissues confirmed the presence of CRC-associated bacteria, such as Fusobacterium and Bacteroides, as well as Propionibacterium allowing discrimination between tumor-associated and contaminant taxa. An internal quality index showed that the degree of similarity within sample pairs inversely correlated with the dominance of contaminant taxa. Given the importance of FFPE specimens for larger studies in human cancer genomics, our findings may provide useful indications on potential confounding factors to consider for accurate and reproducible metagenomics analyses. Keywords: microbiome; colorectal cancer; 16S gene sequencing; RNA in situ hybridization; FFPE Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Oct 29 07:51:53 CEST 2021 ModDate: Fri Oct 29 08:03:04 CEST 2021 ID: 0xd0ca5193cedcb7e9f6cd6154cf1d97fb, 0xd0ca5193cedcb7e9f6cd6154cf1d97fb Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: EHOAYY+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 135 FontDescriptor: FontName: EHOAYY+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JLOVMM+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: JLOVMM+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: 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