15f305a2cb82e9fd58d7dc093a26e096 pmc_10178615.pdf d06e45730e88745a8099a23726fbf6dc9627c13d pmc_10178615.pdf b5604efd7f511980c2b7854aa94726303c7e639548a273e83fce912ce69db4dd pmc_10178615.pdf Title: Characterization of Seminal Microbiome of Infertile Idiopathic Patients Using Third-Generation Sequencing Platform Subject: Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of 16S rRNA gene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the 16S rRNA gene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina’s MiSeq sequencing platform. Comparison between the results obtained with the two platforms has been used to analyze the impact of sequencing method on the study of the seminal microbiome’s composition. Seminal microbiota observed with MinION were mainly composed of the phyla Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria, with the most abundant genera being Peptoniphilus, Finegoldia, Staphylococcus, Anaerococcus, Campylobacter, Prevotella, Streptococcus, Lactobacillus, Ezakiella and Enterococcus. This composition was similar to that found by the Illumina platform, since these 10 most abundant genera were also among the most abundant genera detected by the Nanopore platform. In both cases, the top 10 genera represented more than 70% of the classified reads. However, relative abundance of each bacterium did not correlate between these two platforms, with intraindividual variations of up to 50 percentage points in some cases. Results suggest that the effect of the sequencing platform on the characterization of seminal microbiota is not very large at the phylum level, with slightly variances in Firmicutes and Actinobacteria, but presents differences at the genus level. These differences could alter the composition and diversity of bacterial profiles or posterior analyses. This indicates the importance of conducting multi-platform studies to better characterize seminal microbioma. Keywords: seminal microbiota; MinION; nanopore sequencing; Illumina; male fertility Author: Sergio Garcia-Segura, Javier del Rey, Laia Closa, Iris Garcia-Martínez, Carlos Hobeich, Ana Belén Castel, Francisco Vidal, Jordi Benet and Maria Oliver-Bonet Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Apr 26 07:45:58 2023 CEST ModDate: Wed Apr 26 08:04:27 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2026262 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LOOXHD+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 MHZOCU+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 GHLGHI+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 MFSHPI+VnURWPalladioL Type 1 Custom yes yes no 23 0 YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 46 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 CXSBYQ+URWPalladioL-BoldItal Type 1 Custom yes yes no 71 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 83 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 86 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 89 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 92 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 102 0 KNCPON+PalatinoLinotype CID TrueType Identity-H yes yes yes 105 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 111 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 114 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 117 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 135 0 KNCPON+PalatinoLinotype CID TrueType Identity-H yes yes yes 138 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 144 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 147 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 150 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 175 0 KNCPON+PalatinoLinotype CID TrueType Identity-H yes yes yes 178 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 184 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 187 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 190 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 199 0 KNCPON+PalatinoLinotype CID TrueType Identity-H yes yes yes 202 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 208 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 211 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 214 0 KNDJAM+PalatinoLinotype,Italic TrueType MacRoman yes yes no 228 0 KNCPOM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 231 0 KNCPON+PalatinoLinotype CID TrueType Identity-H yes yes yes 234 0 KNCPOO+PalatinoLinotype TrueType WinAnsi yes yes no 240 0 KNCPPA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 243 0 KNDANM+PalatinoLinotype TrueType MacRoman yes yes no 246 0 ESSRQF+VnURWPalladioL-Italic Type 1 Custom yes yes no 257 0 DKZMYW+CMSY10 Type 1 Builtin yes yes no 264 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 268 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-02 02:35:00 CET RepresentationInformation: pmc_10178615.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-09-08 02:19:18 CEST Size: 2026262 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 469 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Seminal Microbiota Composition Destination: subsection.2.1 Item: Title: Seminal Microbiota Estructure Destination: subsection.2.2 Item: Title: Relative Abundance Comparison between Sequencing Platforms Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Sample Collection Destination: subsection.4.1 Item: Title: Microbiome DNA Extraction Destination: subsection.4.2 Item: Title: 16S rRNA Gene Sequencing Destination: subsection.4.3 Item: Title: Bioinformatics Analysis Destination: subsection.4.4 Item: Title: Statistical Analysis Destination: subsection.4.5 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.A. Info: Title: Characterization of Seminal Microbiome of Infertile Idiopathic Patients Using Third-Generation Sequencing Platform Author: Sergio Garcia-Segura, Javier del Rey, Laia Closa, Iris Garcia-Martínez, Carlos Hobeich, Ana Belén Castel, Francisco Vidal, Jordi Benet and Maria Oliver-Bonet Subject: Since the first description of a commensal seminal microbiome using sequencing, less than a decade ago, interest in the composition of this microbiome and its relationship with fertility has been growing. Articles using next-generation sequencing techniques agree on the identification of the most abundant bacterial phyla. However, at the genus level, there is still no consensus on which bacteria are most abundant in human seminal plasma. This discrepancy may be due to methodological variability such as sample collection, bacterial DNA extraction methodology, which hypervariable regions of 16S rRNA gene have been amplified, or bioinformatic analysis. In the present work, seminal microbiota of 14 control samples and 42 samples of idiopathic infertile patients were characterized based on full-length sequencing of the 16S rRNA gene using MinION platform from Oxford Nanopore. These same samples had been analyzed previously using Illumina’s MiSeq sequencing platform. Comparison between the results obtained with the two platforms has been used to analyze the impact of sequencing method on the study of the seminal microbiome’s composition. Seminal microbiota observed with MinION were mainly composed of the phyla Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria, with the most abundant genera being Peptoniphilus, Finegoldia, Staphylococcus, Anaerococcus, Campylobacter, Prevotella, Streptococcus, Lactobacillus, Ezakiella and Enterococcus. This composition was similar to that found by the Illumina platform, since these 10 most abundant genera were also among the most abundant genera detected by the Nanopore platform. In both cases, the top 10 genera represented more than 70% of the classified reads. However, relative abundance of each bacterium did not correlate between these two platforms, with intraindividual variations of up to 50 percentage points in some cases. Results suggest that the effect of the sequencing platform on the characterization of seminal microbiota is not very large at the phylum level, with slightly variances in Firmicutes and Actinobacteria, but presents differences at the genus level. These differences could alter the composition and diversity of bacterial profiles or posterior analyses. This indicates the importance of conducting multi-platform studies to better characterize seminal microbioma. 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