Impact of different DNA enrichment methods on 16S rRNA amplicon based and nanopore metagenomic sequencing based microbial investigation of low biomass samples

Abstract

The efficacy of DNA sequencing, particularly long reads nanopore sequencing, is critically dependent on the amount and quality of the input DNA. However, extracting high concentrations of DNA from low biomass samples, especially from solid matrices, presents significant challenges, this limitation not only substantially hampers the scope of environmental microbiology studies but also makes enhancing DNA yield indispensable in many instances. Therefore, in this study, we systematically evaluated the impact of four different DNA enrichment methods on both amplicon and metagenomic community analyses of solid-phase, low-biomass samples: permafrost soil and biofilm of sand filter. These methods include multiple displacement amplification (MDA), centrifugal filtration (CF), freeze vacuum drying at (FVD) as well as vacuum centrifugal at 35, 45, and 60 °C (namely VC35, VC45, VC60). Our results indicate that FVD was the most effective for increasing DNA concentration, while VC methods best preserved DNA fragment length. In contrast, the widely used MDA and CF methods exhibited biases, preferentially enriching low-GC content sequences, which affected both assembly and annotation outcomes. Metagenomic assembly from MDA and CF samples was suboptimal, with fewer contigs and no middle quality MAGs recovered compared to other methods. Community composition analysis revealed significant shifts across all enrichment methods, with Sphingomonas and Sphingorhabdus genera could be obviously enriched. These findings highlight the necessity and importance of carefully selecting DNA enrichment methods to ensure reliable metagenomic investigation of low-biomass environmental samples.

Graphical abstract

Keywords

DNA enrichment / Low biomass samples / Nanopore sequencing / Community composition / Metagenomic assembly

Highlight

	● FVD was most effective in increasing DNA concentration in low biomass samples.
	● VC methods best preserved the length of DNA fragments.
	● MDA and CF methods preferentially enrich low-GC sequences.
	● Sphingomonas and Sphingorhabdus could be obviously enriched by all methods.

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