Ultra pure high molecular weight DNA from soil for Nanopore shotgun metagenomics and metabarcoding sequencing

Abstract

Soil microbes are among the most abundant and diverse organisms on Earth but remain poorly characterized. New technologies have made possible to sequence the DNA of uncultivated microorganisms in soil and other complex ecosystems. Genome assembly is crucial for understanding their functional potential. Nanopore sequencing technologies allow to sequence long DNA fragments, optimizing production of metagenome-assembled genomes compared to short-read technology. Extracting DNA with a very high purity and high molecular weight is key to get the most out of this long read technologies. Here we present two extraction protocols to get DNA with high purity. First protocol is optimized to reach DNA quality suiting Nanopore shotgun metagenomics. It uses a non-toxic centrifugation gradient to separate bacterial cells from soil to extract DNA directly on cells. The median length of the acquired DNA sequences (N50) was 3 to 7 times greater than previously published in the literature, achieving an N50 of ∼14 kb. The other, a modification of a commercially available MP Biomedical DNA extraction kit, yielded high-purity DNA for full-length 16S Oxford Nanopore metabarcoding, with an N50 of ∼8 kb. The MP-based protocol achieves higher yields of ultra-pure DNA compared to the Nycodenz protocol, at the expense of shorter fragment lengths.

Keywords: Bacterial cells; Community DNA; Long fragments; MP kit; Metabarcoding; Metagenomics - Oxford nanopore nanopore sequencing; Nycodenz; Solvent free; Ultra Pure HMW DNA extraction and purification from soil for Nanopore sequencing.

Graphical abstract

Fig. 1

Nycodenz based graphical protocol. HMW = high molecular weight. Bp = base pair.

Fig. 2

1 % Agarose gel with DNA samples from different protocols and treatments. 1 – Ultra Pure MP kit based DNA extract 2- Tournier et al. MP kit based DNA ; 3 - Ladder GeneRuler 1 kb+ ; 4 – Nycodenz based DNA extract before RNase treatment; 5 - Nycodenz based DNA extract after RNase treatment (61 µg.µl^-1 37 °C – 30 min) ; 6 – Final product : Nycodenz based Ultra Pure HMW DNA.