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Extraction of Non-Protein Amino Acids from Cyanobacteria for Liquid Chromatography-Tandem Mass Spectrometry Analysis
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Using Repeated Lysis Steps Fractionates Between Heterotrophic and Cyanobacterial DNA Extracted from Xenic
Alexis D Wagner1, Mohammed M A Ahmed2, Victoria Starks1
1Department of BioMolecular Science, School of Pharmacy, University of Mississippi, University, Mississippi, 38677, United States of America.
Biorxiv : the Preprint Server for Biology
|September 4, 2024
Summary
This study optimized DNA extraction from cyanobacteria by using iterative lysis and size selection steps. This method successfully enriched host DNA, enabling complete genome sequencing of two cyanobacteria species.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Cyanobacterial DNA extraction is challenging due to diverse cell structures and common heterotrophic microbiomes.
- Low cyanobacterial DNA yield and high contamination from other species hinder complete genome sequencing.
Purpose of the Study:
- To optimize a DNA isolation protocol for enriching cyanobacterial host DNA from mixed cultures.
- To enable successful genome sequencing of cyanobacteria using nanopore technology.
Main Methods:
- Developed a protocol with three iterative cell lysis steps to reduce microbiome DNA.
- Incorporated two bead-based size selection steps to remove low-molecular-weight DNA fragments.
- Utilized EPI2ME analysis to assess the reduction of heterotrophic microbiome reads.
Main Results:
- Iterative lysis significantly decreased the proportion of heterotrophic microbiome DNA with each step.
- The optimized protocol yielded DNA suitable for nanopore sequencing, overcoming shearing challenges.
- Successfully sequenced and assembled closed chromosomes for *Leptolyngbya* sp. BL-A-14 (7.2 Mb) and *Limnothrix* sp. BL-A-16 (4.5 Mb).
Conclusions:
- The iterative lysis and size selection protocol effectively enriches cyanobacterial DNA for sequencing.
- This method is valuable for sequencing xenic cyanobacterial cultures, where axenic cultures are not feasible.
- The approach also offers potential for studying the associated heterotrophic microbiome composition.
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