Genome assembly and annotation of microalga Nannochloropsis oceanica C018

Microalgae-based technologies have emerged as sustainable approaches to help mitigate the impact of rising CO₂ emissions while providing scalable solutions applicable in agriculture, medicine, and energy, among others. Nannochloropsis microalgae exhibit high productivity and tolerance for elevated CO₂ concentrations, thrive in outdoor conditions, have potential for biofuel production, and have emerged as model organisms for applied phycology (1, 2). Additionally, the biochemical characteristics of Nannochloropsis make them a valuable candidate as a food source for both animals and humans. Nannochloropsis oceanica C018 was isolated by S Brown (University of Hawaii-Manoa) near the Island of Hawaii (19.666° -156.032°) using dilution to extinction with seawater-based F/2 medium (3).

Nannochloropsis oceanica C018 was grown in 20 L carboys with F/2 (3) on a 12-hour day/night light cycle (~400 µmol Q/m²/sec) at 25°C and bubbled with sterile air. Following cell harvesting, DNA was isolated with Quick-DNA/RNA Miniprep Kit D7001 (Zymo Research, USA). Total DNA was quantified using DeNovix DS-11 Series Spectrophotometer/Fluorometer (DeNovix Inc., USA) and normalized to 45 ng/µL prior to sequencing library preparation. DNA fragments between 15 and 20 kb were selected for sequencing, and libraries were prepared using Express Template Prep kit version 3 (Pacific Biosciences, USA) for standard HiFi library preparation. High-fidelity sequencing reads were obtained with Pacific Biosciences Revio system with version SMRT Link v13 and Instrument Control Software (ICS) v13. 1,906,268 total reads were generated with an N50 value of 5090 bp, and HiFi read yield was 11,001,781,445 bp.

Genome assembly followed a modified version of the VGP pipeline (4) in usegalaxy.org (5). Samtools v.1.15.1 (6) was used to extract fastq files from .bam sequence output files, and Cutadapt v.4.6 (7) was implemented to trim adapter sequences and filter low-quality reads. Hifiasm v.0.19.8 (8) was used to assemble contigs from high-fidelity reads with “light” purge level. The resulting assembly was decontaminated with kraken2 v.2.1.1 (9), and duplicates were purged with purge_dups v.1.2.6 (10). Completeness of the resulting genome was estimated with BUSCO v.5.5.0 (11) and quality assessed with QUAST v.5.2.0 (12). Default parameters were used except where otherwise noted.

The resulting genome assembly consisted of 30 contigs, 26.41 Mbp (415× coverage) with a GC content of 54.26%, and a L₅₀ of 10. The genome was annotated following the JGI Annotation pipeline and PhycoCosm (12), with 80 million paired-end transcriptomic reads. Functional annotation of the 8,768 gene models was performed using the JGI annotation pipeline. Both genome assembly size and gene count are within the range of other published Nannochloropsis genomes (13–15) (https://phycocosm.jgi.doe.gov/eustigmatophyta). This genome will spur the continued development of microalgae for use as a biofuel feedstock and provide prerequisite information needed for genetic manipulation and expression analyses.

This work was supported by U.S. Department of Energy grant numbers DE-EE0007091, DE-EE0008518, and DE-EE0009278 and U.S. Department of Agriculture (through Cornell University) grant number 2019-69012-29905 to ZIJ and benefited from the entire “MAGIC” team. This project was also funded by the Duke Bass Connections program. AEG was supported by a U.S./Mexico Fulbright García-Robles award. The work (proposal: 10.46936/10.25585/60008191) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231. Duke Sequencing and Genomic Technologies would like to acknowledge funding from NIH S10OD034222-01 for data obtained with Revio system.