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Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders.
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613926051545088 |
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| access_status_str | Open Access |
| author | Martin, Lauren Courtney |
| author2 | Hemmings, Sian Megan Joanna |
| author_browse | Hemmings, Sian Megan Joanna Martin, Lauren Courtney |
| author_facet | Hemmings, Sian Megan Joanna Martin, Lauren Courtney |
| author_sort | Martin, Lauren Courtney |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128454 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:43:54.041Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128454 Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. Martin, Lauren Courtney Hemmings, Sian Megan Joanna Seedat, Soraya Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Fetal alcohol spectrum disorders Fetus -- Abnormalities -- Complications Vagina -- Diseases Microbial ecology UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Affecting approximately 36% of children in the Western Cape of South Africa, Fetal Alcohol Spectrum Disorder (FASD) describes varying severities of physical, neurodevelopmental, and behavioural deficits associated with prenatal alcohol exposure. Exposure to vaginal microbes during delivery results in the acquisition of intestinal bacteria which, via the microbiome-gut-brain axis, have been found to play a significant role in neurodevelopment. Alcohol-associated vaginal microbial alterations may therefore increase FASD risk in infants. Species-level classifications provide greater insight into the bacterial dynamics of the microbiome. However, attainment of species-level resolution using on-hand hypervariable sequencing data is challenging as information is limited to short regions of the 16S ribosomal RNA (rRNA) gene. We aimed to (1) optimise a novel short-read full-length 16S rRNA amplicon sequencing technique on the Illumina iSeq 100 instrument and (2) perform species-level profiling of the maternal vaginal bacteriome in relation to hazardous alcohol consumption during pregnancy and FASD status of their infants using on-hand targeted V1-V2 hypervariable sequencing data, long-read full-length 16S rRNA amplicon sequencing data from the PacBio Sequel IIe instrument, and data generated from the Illumina iSeq 100. Thereafter, we intended to (3) evaluate the performance of the platforms relative to each other. We hypothesised that several bacterial taxa would be associated with hazardous maternal alcohol consumption during pregnancy and FASD diagnoses in infants. Sequencing data generated by the iSeq 100 was predicted to be the most cost-effective, with data comparable in species-level discriminatory capabilities to the PacBio dataset. Pregnant women (n=28), recruited from antenatal clinics in the Western Cape of South Africa, provided vaginal swab samples on the day of birth. Alcohol use was assessed via AUDIT questionnaire. FASD diagnoses were made by triangulating data from dysmorphology examinations, neurodevelopmental assessments, and maternal interviews. Following amplification, library preparation, and sequencing of 16S rRNA amplicons on the iSeq 100, short-read libraries were re-assembled using a custom-built pipeline, “16S-amplicon-seq”. Microbial composition was also assessed through targeted V1-V2 hypervariable sequencing and long-read 16S rRNA amplicon sequencing on the Illumina MiSeq and the PacBio Sequel IIe instrument, respectively. Microbiome composition and diversity analyses were performed using R packages, dada2, vegan, and phyloseq for all datasets. Associated cost and resultant species-level discrimination were assessed and compared across the three sequencing platforms. The Illumina iSeq 100 sequencing technique generated high-quality data exceeding the expected yield of the platform. Derived near full-length to full-length assemblies were representative of recognised vaginal bacterial taxa. Of the three platforms, long-read sequencing on the PacBio instrument was the most cost-effective and had the highest amplicon sequence variant (ASV) species-level assignments. The iSeq 100’s technique had average performance relative to the other techniques. Significant associations between the abundance of various bacterial genera and species in a number of comparison groups were identified. Additional investigation into the development of cost-effective sequencing techniques with high species-level resolution on instruments with low capital investment is required to improve the accessibility to and usefulness of generated sequencing data. Furthermore, the exploratory nature of this study necessitates replication in a larger cohort, in which true associations between the maternal vaginal microbiota and FASD may be unveiled and used to facilitate the development of novel therapeutic interventions. AFRIKAANSE OPSOMMING: Fetale Alkohol Spektrum Afwyking (FASA) affekteer ongeveer 36% van alle kinders in die Wes-Kaap van Suid-Afrika en is verantwoordelik vir ‘n verskeidenheid van fisiese-, neuro-ontwikkelings-, en gedragsgeoriënteerde afwykings weens voorgeboortelike alkoholblootstelling. Blootstelling aan vaginale mikrobes tydens die geboorteproses lei tot die verkryging van dermbakterieë via die mikrobioom-derm-brein-as en dit speel ‘n noemenswaardige rol in neurologiese ontwikkeling. Gevolglik kan alkohol-geassosieerde vaginale mikrobeveranderinge lei tot ‘n verhoogde risiko van FASA in babas. Klassifikasies op ‘n spesievlak voorsien ‘n groter insig in bakteriële dinamikas in die mikrobioom. Die bereiking van gevolgtrekkings op ‘n spesievlak deur die gebruik van beskikbare hiperveranderlike volgordebepaling is uitdagend aangesien die inligting tot kort streke op die 16S ribosomale RNA (rRNA) geen beperk is. Ons mik om (1) ‘n nuutgevonde kortlesende en vollengte 16S rRNA amplikon volgordebepalingstegniek op die Illumina iSeq 100 instrument te optimisaliseer en (2) spesievlakkige profilering van die vaginale mikrobioom uit te voer na verwysing tot gevaarlike alkoholgebruik gedurende swangerskap en FASA-status van hul babas deur byderhande V1-V2 geteikende hiperveranderlike volgordebepalingsdata, langlesende vollengte 16S rRNA amplikon volgordebepalingsdata van die Pacbio Sequel IIe instrument en data wat van Illumina iSeq 100 gegenereer is. Daarna, beoog ons om (3) verrigting van die platforms relatief tot mekaar te ondersoek. Ons het verwag dat verskeie bakterieë met gevaarlike moederlike alkoholmisbruik en FASA diagnoses in babas geassosieer sou word. Volgordebepalingsdata van die iSeq 100 was voorspel om die mees koste-effektief te wees, met data wat vergelykbaar is met spesievlak diskriminerende vermoëns van die PacBio datastel. Swanger vrouens (n=28) was gewerf vanaf voorboortelike klinieke in die Wes-Kaap van Suid-Afrika, waar vaginaledeppers geneem was op die geboortedag. Alkoholgebruik was getoets deur gebruik te maak van AUDIT. FASA-diagnoses in hulle babas was bepaal deur dismorfologiese ondersoeke, neuro-ontwikkelende ondersoeke, en onderhoude met die moeders te trianguleer. Na amplifisering, voorbereiding van biblioteke en volgordebepaling van die 16S rRNA amplikons op die iSeq 100, was kortlesende biblioteke herversamel deur ‘n pasgemaakte pyplyn, “16S-amplicon-seq”, te benut. Mikrobioom samestellings was ondersoek deur V1-V2 hiperveranderlike-geteikende volgordebepaling en langlesende 16S amplikon volgordebepaling op die MiSeq en PacBio instrumente, afsonderlik, aan te stel. Mikrobioomverwante bioinformatiese samestelling en diversiteitsontleding was uitgevoer vir alle datastelle deur die dada2, vegan, en phyloseq pakkette in R te gebruik. Die verwante koste en gevolglike spesievlakkige diskriminasie was ondersoek en vergelyk oor die volgordebepalingsplatforms. Die Illumina iSeq 100 volgordebepalings tegniek het hoë kwaliteit data, wat die verwagte opbrengs oorskry het, gegenereer. Byna-vollengte tot vollengte versamelings was verteenwoordigend van die herkende vaginale bakteriële taxa. Van die drie platforms, was langlesende volgordebepalings op die PacBio instrument die mees koste-effektief en het ook die hoogste amplikon volgordebepalings-variant (AVV) spesievlakkige toewysings. Die iSeq 100 se tegniek het middelmatige verrigting in vergelyking met die ander tegnieke gehad. Betekenisvolle assosiasies tussen die voorkoms van ‘n verskeidenheid bakteriële genera en spesies, in vele vergelykbare groepe, was geïdentifiseer. Addisionele ondersoeke rondom die ontwikkeling van ‘n koste-effektiewe volgorde bepalings tegniek met hoë spesievlakkige gevolgtrekkings op ‘n instrument met lae-kapitaalbelegging is noodsaaklik om die toegang en bruikbaarheid van goedkoop gegenereerde volgordebepalings data te verhoog. Die ondersoekende natuur van hierdie studie maak replikasie in ‘n groter groep noodsaaklik waarin werklike assosiasies tussen die moederlike vaginale-mikrobioom en FASA onthul en benut kan word om die ontwikkeling van ‘n nuutgevonde terapeutiese ingryping te fasiliteer. Masters 2023-02-27T12:24:53Z 2023-08-30T13:10:32Z 2023-02-27T12:24:53Z 2023-08-30T13:10:32Z 2023-02-27 2023-02-27T12:24:53Z 2023-08-31T09:18:46Z 2023-02-27T12:24:53Z 2023-08-31T09:18:46Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128454 en_ZA Stellenbosch University application/pdf xiii, 159 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Fetal alcohol spectrum disorders Fetus -- Abnormalities -- Complications Vagina -- Diseases Microbial ecology UCTD Martin, Lauren Courtney Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title | Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title_full | Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title_fullStr | Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title_full_unstemmed | Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title_short | Species-level profiling of the maternal vaginal bacteriome using full length 16S rRNA amplicon sequencing with application to Fetal alcohol spectrum disorders. |
| title_sort | species level profiling of the maternal vaginal bacteriome using full length 16s rrna amplicon sequencing with application to fetal alcohol spectrum disorders |
| topic | Fetal alcohol spectrum disorders Fetus -- Abnormalities -- Complications Vagina -- Diseases Microbial ecology UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128454 |
| work_keys_str_mv | AT martinlaurencourtney specieslevelprofilingofthematernalvaginalbacteriomeusingfulllength16srrnaampliconsequencingwithapplicationtofetalalcoholspectrumdisorders |