Abstract
Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.
Highlights
IntroductionLong read sequencing technologies, such as nanopore sequencing, allows for fast, real-time, and culture-free metagenomic sequencing[1]
The nanopore amplification-based library preparation kit (RPB004) uses transposase-mediated cleaving of DNA molecules to attach the primer binding sites for PCR amplification, which should reduce PCR amplification bias. We initially assessed this bias by determining the microbial composition of the ZymoBIOMICS Microbial Community Standard[23] by sequencing using the RPB004 PCR-based library preparation kit and compared the abundance of the different species to the native PCR-free library preparation kit (LSK109)
We used clinical metagenomic samples obtained from vaginal swabs of pregnant women to evaluate the performance to deplete the high content of human DNA that heavily impairs downstream microbiome analyses
Summary
Long read sequencing technologies, such as nanopore sequencing, allows for fast, real-time, and culture-free metagenomic sequencing[1]. In samples containing relatively high proportions of host DNA (> 90%) like saliva, throat, buccal mucosa, and vaginal samples, the detection of low abundant species is expected to be impaired[2]. Host DNA depletion prior to sequencing by selective lysis of host and microbial cells or selective removal of CpG-methylated host DNA are complex wet-lab p rocedures[3]. Host DNA depletion via, e.g., saponin or the “MolYsis Complete5” kit improves the sensitivity of pathogen detection after s equencing[4–6]
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