Technical note: overcoming host contamination in bovine vaginal metagenomic samples with nanopore adaptive sequencing

Abstract

Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium, which was identified at low abundances and categorised under Order “Unclassified Clostridiales” when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations.

Induction of mastitis by cow-to-mouse fecal and milk microbiota transplantation causes microbiome dysbiosis and genomic functional perturbation in mice

“…In this study, more than 8.5 million metagenomic reads were assigned to taxonomic composition which is thought to be sufficient enough to capture maximum microbial richness and corroborated with our previous studies [ 7 , 8 , 12 ]. Several previous studies [ 34 , 35 ] reported that in shotgun metagenome (WMS) study, more than 90% sequence reads may come from host DNA supporting our present findings. One of the hallmark findings of this study is the identification of several new bacterial species associated with both bovine (e.g., L. crispatus , N. pseudobrasiliensis , L. vaginalis , C. difficile , R. insidiosa , B. pseudolongum , Muribaculum sp., Duncaniella sp., D. dubosii ., and Actinoalloteichus sp.)…”

Adaptive sampling during sequencing reveals the origins of the bovine reproductive tract microbiome across reproductive stages and sexes

“…In our study, we adopted ONT long-read technology for adaptive metagenomic sequencing, which reduced the host contamination and enabled non-targeted metagenome identification. The longer sequences also provided more information for taxonomical and functional annotations of the metagenome 22 . The unbiased long-read sequencing approach employed in this study returned an extensive metagenome profile with greater resolution without the hassle of sequencing the sample with different amplicon targets.…”

“…In the past, Ascomycota has only been reported in the bovine reproductive metagenome sequenced using amplicon D1/D2 52 , 53 . Even though ONT adaptive sampling unbiasedly recovered a more extensive metagenomic profile 22 , further investigation shall be conducted to determine and compare its accuracy in recalling metagenomic signatures and relative abundances with other sequencing technologies.…”

“…Adaptive sampling was conducted as described previously 22 . Firstly, the quantity of extracted DNA was measured using Qubit™ 4 fluorometer (Invitrogen™) with Qubit™ dsDNA Broad Range assay kit (Invitrogen™).…”

Seminal and vagino-uterine microbiome and their individual and interactive effects on cattle fertility

“…The 16S rRNA sequencing targets a single gene using primers specific for a hypervariable region, often the V3-V4 region when studying bacteria ( Marotz et al, 2018 ). There is room to introduce bias through primer selection and through possible unequal PCR amplification of the single targeted gene ( Acinas et al, 2005 ; Poretsky et al, 2014 ) which may result in over- or under- expression of certain microbes ( Ong et al, 2022 ). Thanks to advancements in bioinformatic tools and reduced costs, there are other sequencing options such as shotgun metagenomic sequencing that does not target a single gene, but rather retrieves the whole genome in short reads ( Quince et al, 2017 ) and allows the capture of not only the taxonomic identification, but also the function of microorganisms.…”