31c493128c0e75f78c4e78d90014e378 VIEV_a2025v11n1.pdf 4b4b0ac53029c8ac74c34913e247a63bfcf38806 VIEV_a2025v11n1.pdf 73153b2fc0a846d061b277efe6a30689b81a51d6bcc3570faa1e9d18d7b9fe1e VIEV_a2025v11n1.pdf Title: Improvements in RNA and DNA nanopore sequencing allow for rapid genetic characterization of avian influenza Subject: DOI: https://doi.org/10.1093/ve/veaf010 Virus Evolution, 11, 1, xx xx xxxx. Abstract: Avian influenza virus (AIV) currently causes a panzootic with extensive mortality in wild birds, poultry, and wild mammals, thus posing a major threat to global health and underscoring the need for efficient monitoring of its distribution and evolution. We here utilized a well-defined AIV strain to systematically investigate AIV genetic characterization through rapid, portable nanopore sequencing by comparing the latest DNA and RNA nanopore sequencing approaches and various computational pipelines for viral consensus sequence generation and phylogenetic analysis. We show that the latest direct RNA nanopore sequencing updates improve consensus sequence generation, but that the application of the latest DNA nanopore chemistry after reverse transcription and amplification outperforms, such native viral RNA sequencing by achieving higher sequencing accuracy and throughput. We additionally leveraged the direct RNA nanopore sequencing data for the detection of RNA modifications, such as N6-methyladenosine and pseudouridine, which play a role in viral immune evasion. Finally, we applied these sequencing approaches together with portable AIV diagnosis and quantification tools to environmental samples from a poultry farm, demonstrating the feasibility of nanopore sequencing for on-site non-invasive AIV monitoring in real-world outbreak scenarios. Keywords: Avian influenza; viral evolution; RNA modifications; nanopore sequencing Author: Albert Perlas Creator: LuaLaTeX with hyperref package Producer: LuaLaTeX; modified using iTextSharp.LGPLv2.Core 3.7.4.0 CreationDate: Mon Mar 10 12:21:01 2025 CET ModDate: Wed Oct 29 10:39:22 2025 CET Custom Metadata: yes Metadata Stream: yes Tagged: yes UserProperties: no Suspects: no Form: none JavaScript: no Pages: 9 Encrypted: no Page size: 595.276 x 782.362 pts Page rot: 0 File size: 3506475 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IHRKFP+CaeciliaLTStd-Italic CID Type 0C Identity-H yes yes yes 31 0 KKMKWO+CaeciliaLTStd-Roman CID Type 0C Identity-H yes yes yes 30 0 COBZMT+CaeciliaLTStd-Bold CID Type 0C Identity-H yes yes yes 34 0 XVZVYO+LMRoman10-Regular CID Type 0C Identity-H yes yes yes 32 0 LNCUPE+CaeciliaLTStd-Light CID Type 0C Identity-H yes yes yes 33 0 QROFQC+CaeciliaLTStd-Heavy CID Type 0C Identity-H yes yes yes 29 0 TCSEYP+CaeciliaLTStd-LightItalic CID Type 0C Identity-H yes yes yes 35 0 SFAINN+ArialMT TrueType WinAnsi yes yes no 1 0 DTWWFD+LatinModernMath-Regular CID Type 0C Identity-H yes yes yes 36 0 DTWWFD+LatinModernMath-Regular CID Type 0C Identity-H yes yes yes 37 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-10-30 04:14:37 CET RepresentationInformation: VIEV_a2025v11n1.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-10-29 10:39:42 CET Size: 3506475 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Tagged PDF PDFMetadata: Objects: 1947 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: false CenterWindow: false DisplayDocTitle: true NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseOutlines Language: en-US Outlines: Item: Title: Improvements in RNA and DNA nanopore sequencing allow for rapid genetic characterization of avian influenza Children: Item: Title: Introduction Item: Title: Materials and methods Children: Item: Title: Viral diagnosis and quantification Item: Title: Nanopore sequencing Item: Title: Data processing Item: Title: Data subsampling Item: Title: Consensus sequence generation Item: Title: Environmental sample analyses Item: Title: Basecalling of RNA modifications Item: Title: Results Children: Item: Title: DNA- and RNA-based nanopore sequencing of AIV culture Item: Title: Viral consensus sequence generation Item: Title: Viral RNA modifications Item: Title: Nanopore-based AIV genetic characterization from environmental samples Item: Title: Discussion Item: Title: Acknowledgements Item: Title: Author contributions Item: Title: Supplementary data Item: Title: Conflict of interest: Item: Title: Funding Item: Title: Data availability Item: Title: References Info: Title: Improvements in RNA and DNA nanopore sequencing allow for rapid genetic characterization of avian influenza Author: Albert Perlas Subject: DOI: https://doi.org/10.1093/ve/veaf010 Virus Evolution, 11, 1, xx xx xxxx. Abstract: Avian influenza virus (AIV) currently causes a panzootic with extensive mortality in wild birds, poultry, and wild mammals, thus posing a major threat to global health and underscoring the need for efficient monitoring of its distribution and evolution. We here utilized a well-defined AIV strain to systematically investigate AIV genetic characterization through rapid, portable nanopore sequencing by comparing the latest DNA and RNA nanopore sequencing approaches and various computational pipelines for viral consensus sequence generation and phylogenetic analysis. We show that the latest direct RNA nanopore sequencing updates improve consensus sequence generation, but that the application of the latest DNA nanopore chemistry after reverse transcription and amplification outperforms, such native viral RNA sequencing by achieving higher sequencing accuracy and throughput. We additionally leveraged the direct RNA nanopore sequencing data for the detection of RNA modifications, such as N6-methyladenosine and pseudouridine, which play a role in viral immune evasion. Finally, we applied these sequencing approaches together with portable AIV diagnosis and quantification tools to environmental samples from a poultry farm, demonstrating the feasibility of nanopore sequencing for on-site non-invasive AIV monitoring in real-world outbreak scenarios. Keywords: Avian influenza; viral evolution; RNA modifications; nanopore sequencing Creator: LuaLaTeX with hyperref package Producer: LuaLaTeX; modified using iTextSharp.LGPLv2.Core 3.7.4.0 CreationDate: Mon Mar 10 12:51:01 CET 2025 ModDate: Wed Oct 29 10:39:22 CET 2025 Trapped: False ID: 0x306f269e5f3ba2197a0d25d292271db2, 0x990b17f5510ac2b871e791d8c55c2dec Filters: FilterPipeline: FlateDecode Fonts: Type0: Font: BaseFont: XVZVYO+LMRoman10-Regular Encoding: Identity-H ToUnicode: true Font: BaseFont: LNCUPE+CaeciliaLTStd-Light Encoding: Identity-H ToUnicode: true Font: BaseFont: COBZMT+CaeciliaLTStd-Bold Encoding: Identity-H ToUnicode: true Font: BaseFont: TCSEYP+CaeciliaLTStd-LightItalic Encoding: Identity-H ToUnicode: true Font: BaseFont: DTWWFD+LatinModernMath-Regular Encoding: Identity-H ToUnicode: true Font: BaseFont: DTWWFD+LatinModernMath-Regular Encoding: Identity-H ToUnicode: true Font: BaseFont: QROFQC+CaeciliaLTStd-Heavy Encoding: Identity-H ToUnicode: true Font: BaseFont: KKMKWO+CaeciliaLTStd-Roman Encoding: Identity-H ToUnicode: true Font: BaseFont: IHRKFP+CaeciliaLTStd-Italic Encoding: Identity-H ToUnicode: true TrueType: Font: BaseFont: SFAINN+ArialMT FontSubset: true FirstChar: 32 LastChar: 121 FontDescriptor: FontName: SFAINN+ArialMT Flags: Nonsymbolic FontBBox: -664, -324, 2000, 1039 FontFile2: true Encoding: WinAnsiEncoding CIDFontType0: Font: BaseFont: KKMKWO+CaeciliaLTStd-Roman CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: KKMKWO+CaeciliaLTStd-Roman Flags: Symbolic FontBBox: -167, -270, 1136, 926 FontFile3: true Font: BaseFont: COBZMT+CaeciliaLTStd-Bold CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: COBZMT+CaeciliaLTStd-Bold Flags: Symbolic FontBBox: -166, -273, 1141, 938 FontFile3: true Font: BaseFont: XVZVYO+LMRoman10-Regular CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: XVZVYO+LMRoman10-Regular Flags: Symbolic FontBBox: -430, -290, 1417, 1127 FontFile3: true Font: BaseFont: LNCUPE+CaeciliaLTStd-Light CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: LNCUPE+CaeciliaLTStd-Light Flags: Symbolic FontBBox: -164, -266, 1130, 908 FontFile3: true Font: BaseFont: QROFQC+CaeciliaLTStd-Heavy CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: QROFQC+CaeciliaLTStd-Heavy Flags: Symbolic FontBBox: -170, -277, 1146, 970 FontFile3: true Font: BaseFont: TCSEYP+CaeciliaLTStd-LightItalic CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: TCSEYP+CaeciliaLTStd-LightItalic Flags: Symbolic FontBBox: -166, -267, 1033, 909 FontFile3: true Font: BaseFont: DTWWFD+LatinModernMath-Regular CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: DTWWFD+LatinModernMath-Regular Flags: Symbolic FontBBox: -1042, -3060, 4082, 3560 FontFile3: true Font: BaseFont: IHRKFP+CaeciliaLTStd-Italic CIDSystemInfo: Registry: Adobe Registry: Identity Supplement: 0 FontDescriptor: FontName: IHRKFP+CaeciliaLTStd-Italic Flags: Symbolic FontBBox: -166, -270, 1053, 915 FontFile3: true XMP: LuaLaTeX; modified using iTextSharp.LGPLv2.Core 3.7.4.0 1.5 Avian influenza; viral evolution; RNA modifications; nanopore sequencing False application/pdf veaf010_icore_preview.tex Albert Perlas Tim Reska Guillaume Croville Ferran Tarrés-Freixas Jean-Luc Guérin Natàlia Maj00F3$\protect \acute {\text {o}}$ Lara Urban Improvements in RNA and DNA nanopore sequencing allow for rapid genetic characterization of avian influenza Avian influenza; viral evolution; RNA modifications; nanopore sequencing Text OUP Publishing DOI: https://doi.org/10.1093/ve/veaf010 Virus Evolution, 11, 1, xx xx xxxx. Abstract: Avian influenza virus (AIV) currently causes a panzootic with extensive mortality in wild birds, poultry, and wild mammals, thus posing a major threat to global health and underscoring the need for efficient monitoring of its distribution and evolution. We here utilized a well-defined AIV strain to systematically investigate AIV genetic characterization through rapid, portable nanopore sequencing by comparing the latest DNA and RNA nanopore sequencing approaches and various computational pipelines for viral consensus sequence generation and phylogenetic analysis. We show that the latest direct RNA nanopore sequencing updates improve consensus sequence generation, but that the application of the latest DNA nanopore chemistry after reverse transcription and amplification outperforms, such native viral RNA sequencing by achieving higher sequencing accuracy and throughput. We additionally leveraged the direct RNA nanopore sequencing data for the detection of RNA modifications, such as N6-methyladenosine and pseudouridine, which play a role in viral immune evasion. Finally, we applied these sequencing approaches together with portable AIV diagnosis and quantification tools to environmental samples from a poultry farm, demonstrating the feasibility of nanopore sequencing for on-site non-invasive AIV monitoring in real-world outbreak scenarios. en-US 2025-03-10T16:51:01+05:30 © The Author(s) 2025. Published by Oxford University Press. 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