Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples

Abstract

Background: Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms recently implemented in Oxford Nanopore's own control software. By examining the first few hundred bases of a DNA molecule as it passes through a pore, software can determine if the molecule is sufficiently interesting to sequence in its entirety. If not, the molecule is ejected from the pore by reversing the voltage across it, freeing the pore for a new molecule. User supplied sequences define the targets to be sequenced or ejected. Here we explore the potential of using adaptive sampling for enrichment of rarer species within metagenomic samples. Results: We created a synthetic mock community consisting of seven bacterial species at different proportions ranging from 1.2% to 47% and used this as the basis for a series of enrichment and depletion experiments. To investigate the effect of DNA length on adaptive sampling efficiency, we created sequencing libraries with mean read lengths of 1.7 kbp, 4.7 kbp, 10.6 kbp, and 12.8 kbp and enriched or depleted for individual and multiple species over a series of sequencing runs. Across all experiments enrichment ranged from 1.67-fold for the most abundant species with the shortest read length to 13.87-fold for the least abundant species with the longest read length. Factoring in the reduction to sequence output associated with repeatedly rejecting molecules reduces the calculated efficiency of this enrichment to between 0.96-fold and 4.93-fold. We note that reducing ejections due to false negatives (approximately 36%) would significantly increase efficiency. We used the relationship between abundance, molecule length and enrichment factor to produce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlated strongly with experimental data. A web application is provided to allow researchers to explore model predictions in advance of performing their own experiments. Conclusions: Our data clearly demonstrates the benefit for enriching low abundant species in adaptive sampling metagenomic experiments, especially with longer molecules, and our mathematical model can be used to determine whether a given experimental DNA sample is suitable for adaptive sampling. Notably, repeated voltage reversals have no effect on pore stability.

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

“…Nanopore adaptive sampling (NAS) is a recently developed and novel method that performs selective sequencing of individual DNA, cDNA, or RNA molecules in real-time using ONT sequencing technology [22][23][24]. NAS operates through an advanced bioinformatic pipeline that compares nucleotides of individual molecules (~200-400 base pairs) against a userspecified reference file every ~0.4 sec, as sequencing is occurring.…”

“…If present within a biological sample, sequences with a minimum of ~70% sequence similarity to the reference database will be retained [23]. This aspect of NAS distinguishes it from all other sequencing methods, as the process can detect extremely small proportions of non-host DNA/RNA (e.g., low abundance viral and/or bacterial pathogens intermixed with >99% host molecules) [24]. A critical observation with respect to NAS is that the method is not restricted with respect to template length and thus the resulting single-molecule sequences can be thousands of bases long.…”

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

“…This method allows the enrichment or depletion of user specified sequences ( Loose et al, 2016 ; Payne et al, 2020 ). ONT adaptive sampling was demonstrated to be beneficial in enriching low abundant species in a synthetic mock community consisting of 7 bacteria with known proportions ( Martin et al, 2021 ). ONT adaptive sampling can allow deeper sequencing of a metagenome by specifying that the contaminating host DNA be depleted from the sequencing run.…”

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

“…Nanopore adaptive sequencing is a relatively new bioinformatic pipeline (Payne et al 2021), thus comparisons to traditional nanopore sequencing runs are essential. The analyses presented herein and those of others (Gan et al, 2021;Kipp et al, 2021;Martin et al, 2021;Payne et al, 2021;Wanner et al, 2021) (Hlaing et al, 2009);…”