Using Repeated Lysis Steps Fractionates Between Heterotrophic and Cyanobacterial DNA Extracted from Xenic Cyanobacterial Cultures

Abstract

Extracting DNA from cyanobacteria can be a challenge because of their diverse morphologies, challenging cellular structure, and the heterotrophic microbiome often present within cyanobacterial cultures. As such, even when our DNA yields are sufficient for sequencing, the percentage of reads coming from the cyanobacterial host can be low, leading to incomplete genomes spread across several scaffolds. In this research, we optimized a DNA isolation protocol using three iterative cell lysis steps to enrich the portion of DNA isolated coming from the cyanobacterial host rather than the heterotrophic microbiome. In order to utilize in-house nanopore sequencing, we faced a challenge in that our lysis protocol led to DNA shearing and a lower molecular weight DNA extract than is suitable for this sequencing technology. As such we used two bead-based size selection steps to remove shorter molecules of DNA before nanopore sequencing. EPI2ME analysis of the processed reads from the iterative lysis steps showed that in the first lysis the heterotrophic microbiome could make up more than half of all reads, but with each lysis the proportion of reads coming from these other species decreased. Using our iterative lysis protocol, we were able to sequence the genomes of two cyanobacteria isolated from fresh water sources around northern Mississippi, namely Leptolyngbya sp. BL-A-14 and Limnothrix sp. BL-A-16. The genomes of these isolates were assembled as closed chromosomes of 7.2 and 4.5 Mb for Leptolyngbya sp. BL-A-14 and Limnothrix sp. BL-A-16, respectively. Because some cyanobacteria have symbioses with their heterotrophic microbiome it is not always possible to prepare axenic cultures of these organisms, we hope our approach will be useful for sequencing xenic cultures of cyanobacteria, but we can also imagine applications in studying this microbiome specifically by focusing sequencing efforts on the first fraction.

Figure 1.. The iterative lysis workflow.

The initial lysis step is incomplete, after collecting the pellet there is still DNA available to isolate in the solid cell matter. By repeating the lysis steps more vigorously, more DNA can be collected. In addition, because the cyanobacteria are generally more difficult to lyse than heterotrophic bacteria, the proportion of cyanobacterial DNA increases in each lysis fraction.

Figure 2.. The EPI2ME classification of reads from sequencing runs of BL-A-16.

The EPI2ME platform seemed to struggle with classifying cyanobacterial DNA, notice the large fraction of unclassified reads and numerous different cyanobacterial genera annotated for this single species among classified reads. However, the decrease in Pseudomonas reads during the iterative lysis step is clear, dropping from 53% to 17% to 8% of total reads from fraction A to B to C shows clearly the winnowing of heterotrophic DNA from the sample during the iterative lysis.