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DNA isolation methods for Nanopore sequencing of the Streptococcus mitis genome
David Pinzauti1, Francesco Iannelli1, Gianni Pozzi1
1Laboratory of Molecular Microbiology and Biotechnology (LAMMB), Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Abstract:
Streptococcus mitis is a Gram-positive bacterium, member of the oral commensal microbiota, which can occasionally be the etiologic agent of diseases such as infective endocarditis, bacteraemia and septicaemia. The highly recombinogenic and repetitive nature of the S. mitis genome impairs the assembly of a complete genome relying only on short sequencing reads. Oxford Nanopore sequencing can overcome this limitation by generating long reads, enabling the resolution of genomic repeated regions and the assembly of a complete genome sequence. Since the output of a Nanopore sequencing run is strongly influenced by genomic DNA quality and molecular weight, the DNA isolation is the crucial step for an optimal sequencing run. In the present work, we have set up and compared three DNA isolation methods on two S. mitis strains, evaluating their capability of preserving genomic DNA integrity and purity. Sequencing of DNA isolated with a mechanical lysis-based method, despite being cheaper and quicker, did not generate ultra-long reads (maximum read length of 59516 bases) and did not allow the assembly of a circular complete genome. Two methods based on enzymatic lysis of the bacterial cell wall, followed by either (i) a modified CTAB DNA isolation procedure, or (ii) a DNA purification after osmotic lysis of the protoplasts allowed the sequencing of ultra-long reads up to 107294 and 181199 bases in length, respectively. The reconstruction of a circular complete genome was possible sequencing DNAs isolated using the enzymatic lysis-based methods.
Insights
Optimizing DNA isolation for Streptococcus mitis is key for complete genome sequencing using Oxford Nanopore technology. Enzymatic lysis methods yield superior DNA quality, enabling ultra-long reads and full genome assembly.
Area of Science:
- Microbiology
- Genomics
- Bioinformatics
Background:
- Streptococcus mitis, a Gram-positive bacterium, is part of the oral microbiota but can cause serious infections.
- Its repetitive genome structure challenges complete sequencing with short reads.
- Oxford Nanopore sequencing offers long-read capabilities to resolve genomic repeats.
Purpose of the Study:
- To compare three DNA isolation methods for Streptococcus mitis.
- To evaluate DNA integrity and purity for optimal Oxford Nanopore sequencing.
- To determine the best method for achieving complete Streptococcus mitis genome assembly.
Main Methods:
- Three DNA isolation methods were tested on two S. mitis strains: mechanical lysis, enzymatic lysis with CTAB, and enzymatic lysis with osmotic lysis.
- DNA integrity, purity, and read length were assessed.
- Oxford Nanopore sequencing was performed on isolated DNA, followed by genome assembly.
Main Results:
- Mechanical lysis yielded shorter reads (max 59,516 bp) and incomplete genome assembly.
- Enzymatic lysis methods produced ultra-long reads (up to 107,294 bp and 181,199 bp).
- Complete, circular Streptococcus mitis genomes were successfully assembled using DNA from enzymatic lysis methods.
Conclusions:
- Enzymatic DNA isolation methods are superior for preparing Streptococcus mitis DNA for long-read sequencing.
- High-quality DNA is essential for resolving repetitive genomic regions and achieving complete genome assembly.
- These optimized methods facilitate comprehensive genomic studies of Streptococcus mitis.
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