The analysis of long DNA molecules by novel genomic technologies, such as Bionano optical mapping and Third Generation Sequencing, including PacBio Single Molecule Real Time Sequencing and Oxford Nanopore sequencing, provide the opportunity for complete genome characterization and reconstruction, allowing to identify large (balanced) structural variants, to determine the variant phasing and haplotype, to sequence full-length repeated regions and to assemble and scaffold genomes de-novo. Implementation of these technologies requires a combination of highly pure and High Molecular Weight (HMW) DNA, >10^5bp (Bionano Optical Mapping) or >10^4bp (Third Generation Sequencing) in length. However, standardized and suitable extraction methods to obtain highly pure HMW DNA are still missing for many organisms and tissues. In particular, plants and algae store a large amount of phenolic compounds, polysaccharides and a high copy number of chloroplast and mitochondrial DNA, making the extraction of both pure and HMW genomic DNA challenging. The aim of this work was the optimization of methods for the purification of highly pure and (Ultra)HMW DNA from a microalgae selected as case study, Haematococcus pluvialis (H.pluvialis), suitable for Third Generation sequencing and Bionano optical mapping. Despite H.pluvialis is unicellular green microalgae extensively studied for industrial applications, a high quality genome for its biotechnological application is still missing. Therefore, an extensive benchmarking of DNA and nuclei isolation methods was conducted to produce high-quality HMW DNA suitable to generate Third Generation sequencing and Bionano optical mapping data for the reconstruction of its genome de-novo. 4 (U)HMW DNA extraction methods and 8 nuclei isolation methods and 4 post-extraction DNA purification methods were evaluated independently or in combination. To further improve DNA purity and optimize the production of high-quality sequencing data, 4 post-extraction DNA purification methods were also tested. The methods were compared in terms of yield, length and purity of extracted DNA and its analysis by Third Generation sequencing and optical mapping. Only 3 specific combinations of these protocols yielded suitable DNA to generate successful results with PacBio (CTAB buffer+AMPureXP beads purification), Oxford Nanopore (MEB buffer+G-tip- DNA based extraction) and Bionano (MEB buffer+plug- DNA based extraction). The data produced herein can be used to obtain a highly contiguous genome for H.pluvialis with the efficient reconstruction of repetitive genomic portions (highly present in H.pluvialis genome), by eliminating ambiguity in the positions or size of genomic elements.

Optimization of High and Ultra High Molecular Weight DNA purification for Third Generation Sequencing and Optical Mapping in algae

Emanuela Cosentino
2020

Abstract

The analysis of long DNA molecules by novel genomic technologies, such as Bionano optical mapping and Third Generation Sequencing, including PacBio Single Molecule Real Time Sequencing and Oxford Nanopore sequencing, provide the opportunity for complete genome characterization and reconstruction, allowing to identify large (balanced) structural variants, to determine the variant phasing and haplotype, to sequence full-length repeated regions and to assemble and scaffold genomes de-novo. Implementation of these technologies requires a combination of highly pure and High Molecular Weight (HMW) DNA, >10^5bp (Bionano Optical Mapping) or >10^4bp (Third Generation Sequencing) in length. However, standardized and suitable extraction methods to obtain highly pure HMW DNA are still missing for many organisms and tissues. In particular, plants and algae store a large amount of phenolic compounds, polysaccharides and a high copy number of chloroplast and mitochondrial DNA, making the extraction of both pure and HMW genomic DNA challenging. The aim of this work was the optimization of methods for the purification of highly pure and (Ultra)HMW DNA from a microalgae selected as case study, Haematococcus pluvialis (H.pluvialis), suitable for Third Generation sequencing and Bionano optical mapping. Despite H.pluvialis is unicellular green microalgae extensively studied for industrial applications, a high quality genome for its biotechnological application is still missing. Therefore, an extensive benchmarking of DNA and nuclei isolation methods was conducted to produce high-quality HMW DNA suitable to generate Third Generation sequencing and Bionano optical mapping data for the reconstruction of its genome de-novo. 4 (U)HMW DNA extraction methods and 8 nuclei isolation methods and 4 post-extraction DNA purification methods were evaluated independently or in combination. To further improve DNA purity and optimize the production of high-quality sequencing data, 4 post-extraction DNA purification methods were also tested. The methods were compared in terms of yield, length and purity of extracted DNA and its analysis by Third Generation sequencing and optical mapping. Only 3 specific combinations of these protocols yielded suitable DNA to generate successful results with PacBio (CTAB buffer+AMPureXP beads purification), Oxford Nanopore (MEB buffer+G-tip- DNA based extraction) and Bionano (MEB buffer+plug- DNA based extraction). The data produced herein can be used to obtain a highly contiguous genome for H.pluvialis with the efficient reconstruction of repetitive genomic portions (highly present in H.pluvialis genome), by eliminating ambiguity in the positions or size of genomic elements.
High and Ultra High Molecular Weight DNA, DNA extraction, DNA purification, Nuclei Isolation, Third Generation Sequencing, Optical Mapping
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1018425
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