Efficient NGS ready gDNA from microalga v1

Preparing genomic DNA extractions from microalgae specifically for long read sequencing on platforms such as Nanopore and Pac bio requires ultra-high quality DNA free from contaminants that may interfere and inhibit the sequencing chemistry. In microalgae there are many contaminants that can co precipitate with DNA and cause poor run quality on the nanopore platform. Genome sequencing of microalgae can be a difficult task to approach given the microbial consortiums alga are found in. Separating microalgae from bacteria and fungi can be a daunting task and a multifaceted approach is often the most effective. Combining different techniques to obtain pure axenic cultures is often required. The most effective techniques require long regrowth times from single cell colonies, increasing the time required to obtain pure cultures. Many raw read entries into the NCBI SRA are plagued with artefacts from bacteria and fungi. Although sequencing monoclonal axenic cultures is ideal it may not always be practical, by employing density gradient centrifugation, and antibiotic cocktails it is possible to obtain Sequence ready cultures in very short time periods with enough cell mass to produce >5ug of gDNA for nanopore sequencing. The nanopore platform is particularly susceptible to contamination induce failure mode, whatsmore several fungi species have been shown to contain silent contaminants (https://www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life/discussions/awesome-dna-purity-measures-but-quickly-dying-pores?comment=3445) where gDNA preparation test as high quality on the nanodrop ONE platform (thermofisher). Extraction kits are marketed as a one size fits all solution and, in many circumstances, will provide sub standard results and the price of these kits greatly exceeds the face value of the reagents and packaging. Proprietary additives and devices supposedly increase efficiency of extractions and attract a premium, but often perform just as well as a Classic CTAB extraction (carlson). One of the most expensive components of DNA extraction and library preparation are SPRI magnetic beads, which can be easily synthesized in house at a fraction of the cost (BOMB.bio) with the same functionality that enables high performance extractions. To increase the quality of extraction delicate handling and mixing is required, this is best achieved with a rotisserie mixer these have classically had high price tags in order to save costs on this component we 3d printed a model from a recent publication (Karankumar C et. al ). For many situations the ability to customize extractions by varying buffer components and concentrations as well as steps involved can vastly improve the quality of extract and speed up the entire process. By chaining together, a series of extraction and precipitation chemistry into 3 stages, each stage helps remove contaminants that may co-precipitate with other steps. 1. This method exploits the extraction surfactant CTABs ability to only solubilize nucleic acids at high salt concentrations (>1.2M NaCl) where nucleic acids precipitate in a complex with CTAB at a low salt concentration (<0.5M) 2. excess CTAB is then washed out with ethanol and DNA solubilised, cleaned and precipitated with ethanol. 3. Crude DNA is further washed and enzymatically treated before a dual >1kb size selection and PEG based precipitation with DIY-SPRI mix. Each successive step removes more contaminants and improves quality metrics of the extract. Its evident from the nanodrop data there is a hidden contaminent that is not being detected by uv absorbtion spectroscopy. For many first pass extractions (namley KB1 and MUR-279) values appear to be pefectly inrange but then resuspending, cleaning and precipertating with ipa (pass 2) degrades the quality, which is then restored on the third pass with PEG/NaCl precipertation.