## 3 Next-Generation Sequencing Efforts in _Haslea_
### gDNA Extraction  
The HoB4 genome was sequenced through high-throughput short-read Illumina and long-read ONT sequencing platforms. The combination of Illumina and ONT technologies was chosen to take advantage of short-read (better quality) and long-read (better contiguity) sequencing approaches. One clear application for ONT sequencing is de novo genome assembly, in which long reads facilitate the assembly of long contigs. For the generation of long reads on the ONT platform, the DNA needs to be of high molecular weight, and free of contaminants and nicks.  
To achieve this goal, several methods have been tested: different lysis buffers with or without glass beads; different kits; various amounts of the Phenol/Chloroform/Isoamyl alcohol mixture; and RNAse treatment. For some taxa, this is relatively straightforward [51]. However, when experimenting with _Haslea_ spp., the presence of metabolites, including marennine, may pose challenges to the isolation of DNA suitable for various molecular applications. When blue strains of genus _Haslea_ were cultured under high light intensity, they accumulated more marennine compared with the same strains under low light levels [37]. Genomic DNA from cells grown under strong illumination was less amenable to PCR, presumably due to higher accumulation of photosynthetic products and possibly the marennine itself ([PERSON] et al., unpublished observations). If present during the library construction or sequencing on the ONT platform, this may be expected to negatively impact the results. therefore, we extracted gDNA from _Haslea_ cultures grown under low light intensity (50 \(\upmu\)mol photon m\({}^{-2}\) s\({}^{-1}\)) and presenting a reduced accumulation of marennine compared to those grown under standard light intensity (100 \(\upmu\)mol photon m\({}^{-2}\) s\({}^{-1}\)). One widely adopted method of plant DNA isolation entails the inclusion of cetyl trimethyl ammonium bromide (CTAB) in the extraction buffer, which facilitates the separation of carbohydrates from DNA [68]. To enable reliable DNA isolation suitable for genome assembly using the ONT platform for HoB4, we eventually adopted the CTAB-Genomic-tip protocol suggested by ONT for plant leaves [69] with slight modifications, followed by size selection with a Circulomics Short Read Eliminator kit (SKU SS-100-101-01). This final gDNA from HoB4 was used for both ONT and Illumina sequencing. A first aliquot of 7 \(\upmu\)g of HoB4 gDNA was used to prepare the ONT library using the Nanopore Ligation Sequencing Kit (SQK-LSK109). The libraries were sequenced on a MinION Mk1B sequencer on a R9.4.1 flow cell, with a total run time of 96 h. The raw data were base-called with Guppy 4.0.11 [70] in the high-accuracy mode, with the minimum quality score set to 7. Illumina paired-end short-read sequencing was conducted by the Norwegian Sequencing Center (Oslo, Norway), using a second aliquot of HoB4 gDNA. The sequencing outputs are presented in Table 1.  
While nucleic acid extraction was conducted on cell pellets originating from antibiotic/antimycotic-treated cultures of HoB4, the elimination of contaminating microorganisms was incomplete, as indicated by the presence of bacterial reads in the sequencing results (see below).