A comparison between full-length 16S rRNA Oxford nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues

Kenny Yeo; James Connell; George Bouras; Eric Smith; William Murphy; John-Charles Hodge; Suren Krishnan; Peter-John Wormald; Rowan Valentine; Alkis James Psaltis; Sarah Vreugde; Kevin Aaron Fenix

doi:10.1007/s00203-024-03985-7

A comparison between full-length 16S rRNA Oxford nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues

Arch Microbiol. 2024 May 7;206(6):248. doi: 10.1007/s00203-024-03985-7.

Authors

Kenny Yeo^{1

2}, James Connell^{3

4}, George Bouras^{3

4}, Eric Smith^{3

5}, William Murphy^{3

4}, John-Charles Hodge^{3

6}, Suren Krishnan^{3

6}, Peter-John Wormald⁷, Rowan Valentine⁴, Alkis James Psaltis^{3

4}, Sarah Vreugde^{3

4}, Kevin Aaron Fenix^{8

9}

Affiliations

¹ Discipline of Surgery, Adelaide Medical School, The University of Adelaide, Adelaide, SA, 5000, Australia. [email protected].
² Department of Surgery-Otolaryngology Head and Neck Surgery, The University of Adelaide and The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, SA, 5000, Australia. [email protected].
³ Discipline of Surgery, Adelaide Medical School, The University of Adelaide, Adelaide, SA, 5000, Australia.
⁴ Department of Surgery-Otolaryngology Head and Neck Surgery, The University of Adelaide and The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, SA, 5000, Australia.
⁵ Department of Haematology and Oncology, Basil Hetzel Institute for Translational Health Research and The Queen Elizabeth Hospital, Central Adelaide Local Health Network, Adelaide, SA, 5000, Australia.
⁶ Department of Otolaryngology, Head and Neck Surgery, Royal Adelaide Hospital, Adelaide, SA, 5000, Australia.
⁷ Department of Surgery-Otolaryngology Head and Neck Surgery, The University of Adelaide, Adelaide, SA, 5000, Australia.
⁸ Discipline of Surgery, Adelaide Medical School, The University of Adelaide, Adelaide, SA, 5000, Australia. [email protected].
⁹ Department of Surgery-Otolaryngology Head and Neck Surgery, The University of Adelaide and The Basil Hetzel Institute for Translational Health Research, Central Adelaide Local Health Network, Adelaide, SA, 5000, Australia. [email protected].

Abstract

Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R² = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.

Keywords: 16S ribosomal RNA; Head and neck cancer; Long read sequencing; Microbiome.

Publication types

Comparative Study

MeSH terms

Adult
Aged
Bacteria* / classification
Bacteria* / genetics
Bacteria* / isolation & purification
Female
Head and Neck Neoplasms* / genetics
Head and Neck Neoplasms* / microbiology
High-Throughput Nucleotide Sequencing
Humans
Male
Microbiota / genetics
Middle Aged
Nanopore Sequencing* / methods
Phylogeny
RNA, Ribosomal, 16S* / genetics
Sequence Analysis, DNA
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods

Substances

RNA, Ribosomal, 16S