Method for isolation of high molecular weight genomic DNA from Botryococcus biomass

Ivette Cornejo-Corona; Devon J. Boland; Timothy P. Devarenne

doi:10.1371/journal.pone.0301680

Abstract

The development of high molecular weight (HMW) genomic DNA (gDNA) extraction protocols for non-model species is essential to fully exploit long-read sequencing technologies in order to generate genome assemblies that can help answer complex questions about these organisms. Obtaining enough high-quality HMW gDNA can be challenging for these species, especially for tissues rich in polysaccharides such as biomass from species within the Botryococcus genus. The existing protocols based on column-based DNA extraction and biochemical lysis kits can be inefficient and may not be useful due to variations in biomass polysaccharide content. We developed an optimized protocol for the efficient extraction of HMW gDNA from Botryococcus biomass for use in long-read sequencing technologies. The protocol utilized an initial wash step with sorbitol to remove polysaccharides and yielded HMW gDNA concentrations up to 220 ng/μL with high purity. We then demonstrated the suitability of the HMW gDNA isolated from this protocol for long-read sequencing on the Oxford Nanopore PromethION platform for three Botryococcus species. Our protocol can be used as a standard for efficient HMW gDNA extraction in microalgae rich in polysaccharides and may be adapted for other challenging species.

Citation: Cornejo-Corona I, Boland DJ, Devarenne TP (2024) Method for isolation of high molecular weight genomic DNA from Botryococcus biomass. PLoS ONE 19(7): e0301680. https://doi.org/10.1371/journal.pone.0301680

Editor: Susmita Lahiri (Ganguly), University of Kalyani, INDIA

Received: September 19, 2023; Accepted: March 19, 2024; Published: July 24, 2024

Copyright: © 2024 Cornejo-Corona et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. B. alkenealis and B. lycopadienor sequence data related to this study can be found under NCBI BioProject: PRJNA1079018.

Funding: This work was supported by funds from the Texas A&M Department of Biochemistry & Biophysics to T.P.D. D.J.B. was supported by funding from the G. Rollie White Trust, Texas A&M University College of Agriculture & Life Sciences Endowed Graduate Fellowship for the Borlaug Scholars Program. Portions of this research were conducted with the advanced computing resources provided by Texas A&M High Performance Research Computing.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The latest long-read DNA sequencing technologies have the potential to generate data to help answer genomics-based questions that were previously difficult to address due to problems producing long sequencing reads from DNA fragments of 10 kb or longer [1]. The ability to generate such sequencing data enables the assembly of genome sequences to allow researchers to categorize genes, biosynthetic pathways, and even entire organisms more accurately [2, 3]. Long-read sequencing demands high purity, high molecular weight (HMW) genomic DNA (gDNA) of ≥20 kb [4], which can be challenging for non-model species. Thus, applying these sequencing technologies to non-model organisms requires tailoring and optimizing protocols for isolation of HMW gDNA.

Microalgae are organisms that produce molecules with potential applications to many industries, making them of high scientific interest [5, 6]. However, a challenge for their industrial application is achieving optimal yields and productivity of the molecule(s) of interest in wild-type strains [7]. Genetic engineering could play a decisive role in overcoming such limitations [8, 9]. The recent availability of good quality algal genome sequences together with other omics datasets offer information to guide strategic engineering for microalgae strain improvement related to synthetic biology applications [10–12]. Particularly interesting green microalgal species are those in the genus Botryococcus, which can naturally accumulate acyclic hydrocarbons in the range of 30 to 60% of dry weight [13–15]. Historically, Botryococcus was classified as a single species, B. braunii, divided into three chemical races, A, B, and L, that were defined based on the type of hydrocarbon produced. Recently, we used genome assemblies to show that these races are actually three separate species and have been renamed B. alkenealis (formerly A race), B. braunii (formerly B race), and B. lycopadienor (formerly L race) [16].

These Botryococcus species are colonial and the hydrocarbons biosynthesized by these species are produced inside cells and exported into an extracellular matrix (ECM) that holds the cells of the colony together [17, 18]. The ECM is made up of cross-linked long chain aliphatic aldehydes, and the colony is surrounded by a polysaccharide sheath consisting of 2–3 μm long fibrils [18–21]. The three species of Botryococcus are primarily classified according to the type of hydrocarbons they produce; B. alkenealis produces C₂₃-C₃₃ alkadienes/alkatrienes, B. braunii produces C₃₀-C₃₇ triterpenes named botryococcenes, and B. lycopadienor produces the C₄₀ tetraterpene called lycopadiene [21–23]. All of these hydrocarbons can be readily converted into petroleum-equivalent transportation fuels such as gasoline, jet fuel, and diesel [15]. Thus, Botryococcus species could be used as a source of feedstocks for the production of renewable combustion engines fuels. Despite the potential and interest in these Botryococcus species, the biosynthetic pathways and molecular mechanisms for hydrocarbon biosynthesis have not yet been fully described or understood, partly due to the lack of genome assemblies. This deficit has been addressed with our recent genome assemblies for B. alkenealis and B. lycopadienor [16] along with our earlier B. braunii genome assembly [24].

The B. alkenealis and B. lycopadienor genome assemblies [16] utilized a new HMW gDNA isolation protocol we developed that is optimized for Botryococcus biomass. The presence of high amounts of polysaccharides in the ECM of Botryococcus [18, 25] presents a significant challenge to obtaining a high yield of HMW gDNA, and the abundance of hydrocarbons complicates the phase separation during conventional gDNA extraction methods. Prior to developing the optimized method, we attempted to extract HMW gDNA from Botryococcus biomass using different commercial kits, extraction buffer formulations, and lysis conditions, but none of these approaches yielded HMW gDNA suitable for long-read sequencing. Thus, we developed a HMW gDNA extraction protocol to ensure high yield, quality, and integrity of the HMW gDNA from Botryococcus biomass. For example, we integrated a pretreatment step with sorbitol to remove ECM polysaccharides [26] and the brief use of sonication to properly homogenize the samples with minimal disruption of the HMW gDNA. To ensure purity, we also included PVP-40 which removes polyphenolic compounds that are often co-precipitated with nucleic acids [27], a standard RNase treatment, and a long incubation step for nucleic acid precipitation [28]. The resulting optimized protocol is an efficient method suitable for long-read sequencing of Botryococcus HMW gDNA [16].

Materials and methods

The protocol described in this peer-reviewed article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io.j8nlkoz2xv5r/v1, and is included for printing as S1 File with this article.

Botryococcus culturing and biomass preparation

B. alkenealis, (Yamanaka strain [29]), B. braunii (Showa or Berkeley strain [30]), and B. lycopadienor (Songkla Nakarin strain [31]) were cultured in 1 L roux flasks containing 750 ml of modified Chu 13 medium pH 7.5 at 22°C under continuous aeration with filter-sterilized air enriched with 2.5% CO₂. The cultures were maintained for 6 weeks under a 12:12 hours light:dark cycle with a light intensity of 280 μmol photons/m²/s. The modified Chu 13 medium contained the following chemical concentrations: KNO₃ (0.4 g/L), MgSO₄-7H₂O (0.1 g/L), K₂HPO₄ (0.052 g/L), CaCl₂-2H₂O (0.054 g/L), FeNa-EDTA (0.01 g/L), H₃BO₄ (2.86 mg/L), MnSO₄-H₂O (1.54 mg/L), ZnSO₄-7H₂O (0.22 mg/L), CuSO₄-5H₂O (0.08 mg/L), NaMoO₄-2H₂O (0.06 mg/L) and CoSO₄-7H₂O (0.09 mg/L). 40-day-old fresh biomass from cultures in the stationary phase was harvested by filtration using a 10 μm nylon net, the biomass immediately frozen with liquid N₂, and stored at -80°C.

Buffer preparation

The sorbitol wash buffer contained 100 mM Tris-HCl pH 8.0, 0.35 M sorbitol, 5 mM EDTA pH 8.0, 1% (W/V) polyvinylpyrrolidone 40,000 MW (PVP-40), and 1% (V/V) 2-mercaptoethanol (β-ME). The DNA extraction buffer contained 100 mM Tris-HCl pH 8.0, 3 M NaCl, 3% cetyltrimethyl ammonium bromide (CTAB), 20 mM EDTA, 1% (W/V) PVP-40, and 1% (V/V) β-ME. Each of these buffers was prepared without β-ME, autoclaved, stored at 4°C, and the β-ME was added just prior to use. TE buffer contained 10mM Tris-HCl and 1mM EDTA pH 8.0. The CHCl₃:IAA wash buffer contained a 24:1 mixture of chloroform:isoamyl alcohol and was stored at 4°C.

DNA extraction protocol

The HMW DNA extraction protocol is summarized in Fig 1 and S1 File. Initial tests indicated optimal HMW gDNA extraction was obtained using between 100–110 mg of liquid N₂ ground biomass. In step 1 (see Fig 1), biomass is harvested, or previously harvested biomass is removed from the -80°C freeze, the biomass ground in a mortar and pestle with liquid nitrogen, 100–110 mg samples quickly weighed to minimize thawing, samples transferred to 1.5 mL eppendorf tubes, and samples kept frozen with liquid nitrogen.

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Fig 1. Workflow for extraction of HMW gDNA from Botryococcus.

Created with BioRender.com.

https://doi.org/10.1371/journal.pone.0301680.g001

In step 2 (see Fig 1), one ml of sorbitol wash buffer was added to each sample, the samples allowed to thaw while resuspending the biomass by vortexing (Fig 2A–2C), and samples were sonicated on ice for 25 seconds using a tip sonicator (Sonic Dismembrator, Fisher Scientific, model 100) at 30% power. Next, samples were centrifuged at 2,500 x g at room temperature (RT) for 5 minutes, and the liquid phase carefully removed, avoiding disruption of the floating and pelleted biomass layers formed during centrifugation (see Fig 2D–2F). The liquid phase was discarded and the sorbitol wash repeated a total of three times removing and discarding the liquid phase each time. The floating and pelleted biomass was saved and used for HMW gDNA extraction in the next step.

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Fig 2. Images of ground Botryococcus biomass in sorbitol wash buffer before and after centrifugation.

(A), (B), (C) Homogenized biomass resuspended in sorbitol wash buffer before centrifugation for each Botryococcus species. (D), (E), (F) Separation of biomass phases in sorbitol wash buffer after centrifugation. f, floating biomass phase; s, liquid supernatant; p, biomass pellet phase. The f and p phases are saved after each wash step.

https://doi.org/10.1371/journal.pone.0301680.g002

To start the HMW gDNA extraction in step 3 (see Fig 1), 700 μl of DNA extraction buffer (pre-warmed at 65°C) was added to each sample containing the floating and pelleted biomass, the samples resuspended by vortexing, samples incubated at 65°C for 30 minutes with mixing by inversion every 10 min, and samples incubated at RT for 5 minutes. Next, 700 μl of CHCl₃:IAA buffer was added to each sample, samples vigorously mixed by vortexing for 10 seconds, samples centrifuged at 2,500 x g for 10 min at RT, and the upper aqueous phase transferred to a new 1.5 ml eppendorf tube being careful not to disturb of the debris at the phase interface. This resulted in recovery of approximately 500 μl of the aqueous phase that was then placed on ice.

In step 4 (see Fig 1), the RNA was removed by adding 2 μl of RNase A (25 mg/ml) to each sample to a final concentration of 0.1 mg/ml, the samples incubated at 37°C for 15 minutes with mixing by inversion every 5 minutes, 500 μl of CHCl₃:IAA wash buffer was added to each sample, the samples mixed by vortexing for 10 seconds, the samples centrifuged at 13,000 x g for 10 minutes at 4°C, the upper phase was transferred to a fresh tube, and samples placed on ice.

In step 5 (see Fig 1), the HMW gDNA was precipitated by adding 0.1 volumes of 3M sodium acetate pH 5.2 and 0.66 volumes of cold (-20°C) isopropanol. Samples were mixed by inversion and incubated at -20°C overnight. Following incubation, samples were centrifuged at 13,000 x g for 10 minutes at 4°C, the supernatant removed by decanting, and tubes were left to drain by resting inverted on paper towels at RT until the samples were dry.

In step 6 (see Fig 1), the dried pellets were washed by the adding 1 ml of cold (-20°C) 70% ethanol, samples mixed by inversion, the samples centrifuged at 13,000 x g for 10 minutes at 4°C, the supernatants removed by aspiration trying avoid loss or disturbance of DNA pellet, and the samples dried using a vacuum centrifuge for 10 minutes at 36°C. Finally, the HMW gDNA was resuspended by adding 100 μl TE buffer, incubated for 10 minutes at RT without pipetting to minimize DNA shearing, the samples gently homogenized by inversion, and 50 μl aliquots stored at -80°C until used.

Qualitative and quantitative analysis of isolated HMW gDNA

Approximately 200 ng of each HMW gDNA sample was analyzed by electrophoresis using a 0.5% agarose gel in Tris-acetate EDTA buffer (TAE) with 0.5 μg/ml ethidium bromide and molecular weight markers (Quick-Load 1 kb Extend DNA Ladder, NEB). Electrophoresis was performed for 15 minutes at 120 volts, and visualization was done using a UV transilluminator for a qualitative check of the abundance, size distribution, and quality for all samples. For a quantitative analysis of the HMW gDNA samples, 230 nm, 260 nm, and 280 nm absorbance readings were taken using a Biotek Epoch spectrophotometer, the DNA concentration calculated using the A₂₆₀ reading, and A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios calculated for assessment of protein and polysaccharide contamination, respectively.

HMW gDNA size distribution analysis

The HMW gDNA samples from each Botryococcus species were sent to the sequencing facility at Cold Spring Harbor Laboratory (CSHL) for long-read sequencing. Prior to sequencing, CSHL analyzed each sample for fragment size distribution of the isolated HMW gDNA using an Agilent Femto Pulse system.

Long-read sequencing

Long-read sequencing was carried out at the Cold Spring Harbor Laboratory (CSHL) Sequencing Technologies and Analysis core facility using the Oxford Nanopore PromethION platform. HMW gDNA libraires for each Botryococcus species were multiplexed on a single PromethION flow cell. Raw sequencing data was then base called and demultiplexed using Guppy v4. In total, two flow cells were run due to complications with the quality of the sequencing data on the first flow cell. The flow cell that produced lower quality data will be referred to as flow cell 1, and the flow cell with higher quality data referred to as flow cell 2. Briefly, both sets of reads from each flow cell were subjected to the same pre-assembly trimming process, however flow cell 1 had sufficiently less reads post-trim than flow cell 2, reflecting an overall lower quality of the reads obtained. The data from both flow cells were used in the final genome assemblies for B. alkenealis and B. lycopadienor [16]. However, the read summary analysis presented here (Table 1) depicts only flow cell 2 reads to present an “ideal” outcome from this method of HMW gDNA isolation. Sequencing read metrics were analyzed using a custom script (S2 File). Reads were ordered by length from largest to smallest, and standard metrics such as N50/90 BP and N50/90 NUM were determined (Table 1).

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Table 1. Raw sequencing metrics obtained on the Oxford Nanopore PromethION platform from a single multiplexed flow cell run.

Number of Reads: total number of reads obtained; Total BP: total number of base pairs sequenced; Max Read Size: size of the longest read in bp; N50 BP: total length in bp that equals 50% of the Total BP; N50 NUM: number of reads used to obtain the N50 BP value; N90 BP: total length in bp that equals 90% of the Total BP; N90 NUM: number of reads used to obtain the N90 BP value; MEAN: mean read length, MEDIAN: median read length; Coverage: ratio of Total BP to genome size.

https://doi.org/10.1371/journal.pone.0301680.t001

Results and discussion

Rationale for developing new HMW gDNA extraction protocol for Botryococcus species

In order to obtain HMW gDNA from the Botryococcus species B. alkenealis, B. braunii, and B. lycopadienor we optimized an extraction protocol able to remove polysaccharides while resulting in sufficient quantities of highly pure HMW gDNA of the largest sizes possible and suitable for long-read sequencing platforms. Specifically, we aimed to achieve A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios greater than 1.8, and as much HMW gDNA as possible with fragments sizes of 50 kb or higher [6]

Extracting HMW gDNA from Botryococcus species has been a challenge due to the unique physical characteristics of Botryococcus colonies and the presence of large amounts of polysaccharides, which leads to inefficient HMW gDNA extraction [26, 32]. Since sorbitol has been established as an effective pretreatment for removing excess polysaccharides from polysaccharide-rich plant tissues [26], we used a sorbitol-based wash buffer after biomass maceration to ensure the removal of polysaccharides in our samples (Fig 1, step 2). Additionally, the CTAB-based extraction buffer used in our protocol (Fig 1, step 3) allows for the removal of any remaining polysaccharides during extraction of HMW gDNA [33–35]. As outlined below, this approach proved to be efficient in removing polysaccharides, resulted in relatively high recovery of HWM gDNA, and was suitable for long-read sequencing. However, the recovery of HMW gDNA ≥50 kb was low, especially for B. braunii.

Overview of HMW gDNA extraction process

The HMW gDNA extraction protocol is outlined in Fig 1 and S1 File and will be briefly described here. Not all steps or details are discussed below and the Material and Methods section and S1 File should be used to obtain step by step details. Step 1 of the protocol involves harvesting, freezing, and grinding the Botryococcus biomass. We use a nylon mesh with a 10 μm cutoff placed over a Buchner funnel to filter the water from the biomass. We prefer the nylon mesh because the flexibility allows for easy collection of the biomass from the filter. Once filtering is complete, a rubber spatula is used to remove a small amount of biomass from the mesh, which is immediately placed in a 50 ml Falcon tube containing liquid nitrogen. This is repeated until all biomass is removed from the filter, collected into a single Falcon tube, and the Falcon tube is placed in the -80°C freezer until needed. This process allows for many small clumps of biomass to be collected into a single tube that can be easily removed for grinding without removing all the biomass. The grinding of the biomass follows standard procedures for using a mortar and pestle with liquid nitrogen. Once ground, weighing of aliquots should be done as quickly as possible to minimize thawing. Samples are transferred to eppendorf tubes and returned to liquid nitrogen.

In step 2 (Fig 1), the majority of the polysaccharides are removed with a sorbitol wash buffer that is added to the ground tissue and the samples should be homogenized in the buffer and thawed at this point (Fig 2A–2C). The polysaccharides are removed by centrifugation to separate solid biomass from the polysaccharides dissolved in the liquid phase. Since Botryococcus cells/colonies contain high amounts of hydrocarbons, the biomass containing hydrocarbons floats to the top of the liquid phase and biomass without hydrocarbons is pelleted (Fig 2D–2F). The liquid supernatant phase containing polysaccharides should be removed and discarded, leaving behind the floating and pelleted biomass. This process is repeated three times and after each wash the size of the pelleted biomass will increase.

In step 3, the DNA is extracted from the floating and pelleted biomass using an extraction buffer, which contains CTAB since it can remove any remaining polysaccharides and PVP-40 that removes polyphenolic compounds that are often bound to and co-precipitated with nucleic acids [33–35]. This is followed by a standard wash with chloroform:isoamyl alcohol (CHCl₃:IAA), saving the aqueous phase for step 4.

The remaining parts of the protocol follow standard steps for DNA extractions: In step 4, the RNA in the sample is digested followed by a CHCl₃:IAA wash, the DNA is precipitated with sodium acetate and isopropanol in step 5, and in step 6 the precipitated DNA is washed with ethanol, dried, and resuspended in TE buffer.

Qualitative and quantitative analysis of HMW gDNA

The HMW gDNA isolated from each Botryococcus species was qualitatively analyzed by running approximately 200 ng of each sample on a 0.5% agarose gel. This analysis suggests that HMW gDNA was successfully isolated from each species with the B. alkenealis and B. lycopadienor samples having the largest molecular weight sizes of HMW gDNA followed by B. braunii (Fig 3A). The smear of lower molecular weight gDNA in the B. braunii sample (Fig 3A) may indicate enrichment of smaller gDNA fragments in this sample.

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Fig 3. Qualitative and quantitative analysis of HMW gDNA extracted from the three Botryococcus species.

(A) 0.5% agarose gel electrophoresis of 200 ng HMW gDNA from each Botryococcus species. (B) DNA concentration based on A₂₆₀ analysis. (C) A_260/230 ratio to assess polysaccharide contamination. (D) A_260/280 ratio to assess protein contamination. In (C) and (D), data represents the mean ± standard error of four independent samples for B. alkenealis and B. lycopadienor, and six samples for B. braunii. A one-way ANOVA with the Friedman test was performed, and no significant differences were found between samples. (E) Size distribution of extracted HMW gDNA using an Agilent Femto Pulse system. Vertical dashed line indicates HMW gDNA ≥50kb in each sample.

https://doi.org/10.1371/journal.pone.0301680.g003

Each sample was then analyzed by absorbance spectroscopy at 260 nm (nucleic acids), 230 nm (polysaccharides), and 280 nm (protein) in order to quantitate the HMW gDNA recovery and estimate polysaccharide and protein contamination. The recovery of HMW gDNA ranged from 150 ng/μl to 225 ng/μl (Fig 3B), while the A₂₆₀/A₂₈₀ (Fig 3C) and an A₂₆₀/A₂₃₀ (Fig 3D) ratios were at 1.8 or higher for each sample indicting sufficient removal of polysaccharides and proteins. This data was obtained from four individual samples for B. alkenealis and B. lycopadienor and six samples for B. braunii. The samples for each species were pooled for the analysis in Fig 3. The raw data for each sample is shown in S1 Table.

Size distribution of isolated HMW gDNA

The size distribution of the extracted HMW gDNA was further analyzed using the Agilent Femto Pulse System, which allows for more accurate analysis of DNA fragment size distribution as compared to agarose gel electrophoresis. The results showed that the isolated gDNA fragments in each sample spanned a large size range between 1.3 kb and 165 kb (Fig 3E). For each species the majority of gDNA fragments were in the 10 kb to 50 kb range, with B. braunii have a much lower amount of total gDNA fragments and a large amount of fragments in the 850 bp range (Fig 3E). Having a large portion of gDNA fragments of at least 10 kb in each sample (Fig 3E) is particularly important since typical long-read libraries require an insert size ranging from 500 bp to greater than 20 kb to obtain raw reads of 10 kb on average [4]. The proportion of HMW gDNA fragments recovered that were ≥50 kb was relatively small and was best for B. lycopadieneor followed by B. alkenealis and B. braunii (Fig 3E). This analysis suggests that the quality of the HMW gDNA isolated from B. lycopoadienor had the highest quality in terms of fragment length followed by B. alkenealis and B. braunii having poor quality.

Raw sequencing metrics obtained from the isolated HMW gDNA

The purified HMW gDNA was sequenced using the Oxford Nanopore PromethION platform and was used for genome assembly of the three Botryococcus species. The details of how this data was processed into genome assemblies is presented in our genome analysis study for each of the Botryococcus species [16]. An analysis of the metrics obtained from these sequences are presented here and the data are shown in Table 1. The sequencing resulted in the generation of 372,000, 540,000, and 468,000 total sequence reads with N50s of 0.3 kb, 0.4 kb, and 0.4 kb, and a mean read length of 5.1 kb, 2.7 kb, and 3.8 kb for B. alkenealis, B. braunii, and B. lycopadienor, respectively (Table 1). Based on the predicted genome sizes for each Botryococcus species [24, 36], the sequence reads had genome coverages of 10.26x, 8.7x, and 13.2x, for B. alkenealis, B. braunii, and B. lycopadienor, respectively, indicating high sequencing depth. The sequences for B. braunii had the most number of sequence reads but the lowest total base pairs, mean sequence length, and genome coverage (Table 1). This suggest most of the sequence reads for B. braunii were short and supports the HMW gDNA size distribution analysis shown in Fig 3E.

Conclusions

Developing efficient protocols for HMW gDNA extraction is indispensable to achieve good quality genome assemblies [3, 37]. Traditionally, short-read sequencing lacks the sequence length to cover complex regions within genomes such as those in plants that have large, repeat-rich genomes [38]. Long-read sequencing can overcome these problems by bridging these complex sequence regions within genomes [1]. Thus, we developed a HMW gDNA extraction protocol to isolate gDNA from Botryococcus species suitable for long-read sequencing.

The new HMW gDNA extraction method we developed removes the polysaccharides associated with Botryococcus species, resulting in HMW gDNA of high purity, in higher concentrations than previously published protocols [32], and with a large portion of the gDNA fragment sizes isolated being 10 kb or larger. Thus, this HMW gDNA was suitable for long-read sequencing platforms, and sequencing on the Oxford Nanopore PromethION platform resulted in long sequence reads suitable for genome assembly. The long-read sequences generated were used for the de novo genome assembly for B. alkenealis and B. lycopadienor [16]. However, the HMW gDNA and resulting long-read sequences from B. braunii were of lower quality and length, which did not allow us to improve on our previous B. braunii genome assembly [24]. It is not clear at this time why the HMW gDNA from B. braunii was of lower yield and quality as compared to B. alkenealis and B. lycopadienor. It will be interesting to see if this optimized method is suitable for the extraction of HMW gDNA from other microalgae species that have a high polysaccharide content.

Supporting information

S1 Table. Raw quantitation data for each HMW gDNA sample isolated from the three Botryococcus species.

https://doi.org/10.1371/journal.pone.0301680.s001

(PDF)

S1 File. Step-by-step protocol for HMW gDNA extraction.

https://doi.org/10.1371/journal.pone.0301680.s002

(PDF)

S2 File. PDF document with custom script for analyzing sequence reads.

https://doi.org/10.1371/journal.pone.0301680.s003

(PDF)

Acknowledgments

Portions of this research were conducted with the advanced computing resources provided by Texas A&M High Performance Research Computing.

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Figures

Abstract

Introduction

Materials and methods

Botryococcus culturing and biomass preparation

Buffer preparation

DNA extraction protocol

Qualitative and quantitative analysis of isolated HMW gDNA

HMW gDNA size distribution analysis

Long-read sequencing

Results and discussion

Rationale for developing new HMW gDNA extraction protocol for Botryococcus species

Overview of HMW gDNA extraction process

Qualitative and quantitative analysis of HMW gDNA

Size distribution of isolated HMW gDNA

Raw sequencing metrics obtained from the isolated HMW gDNA

Conclusions

Supporting information

S1 Table. Raw quantitation data for each HMW gDNA sample isolated from the three Botryococcus species.

S1 File. Step-by-step protocol for HMW gDNA extraction.

S2 File. PDF document with custom script for analyzing sequence reads.

Acknowledgments

References