Improved genome assembly of Aspergillus niger ATCC 1015, a citric acid-producing isolate

ABSTRACT

We present a high-quality hybrid genome assembly of Aspergillus niger ATCC 1015, a citric acid-producing isolate. The genome consists of eight nuclear chromosomes with a length of 35,617,925 bp (N50: 4.3 Mb; GC: 49.6%) and a 31,333 bp mitochondrial genome, providing a valuable resource for biotechnological research and applications.

ANNOUNCEMENT

Aspergillus niger ATCC 1015 is an industrial strain used for citric acid production (1). Here, we present a new hybrid Nanopore-Illumina genome assembly of this strain, improving contiguity over the prior versions (2, 3). This resource enhances the genetic foundation and supports strain engineering for biotechnological applications.

Aspergillus niger ATCC 1015, obtained from the Westerdijk Fungal Biodiversity Institute (CBS 113.46) in April 2025, was passaged twice prior to sequencing on minimal medium (1.5% agar, 1% glucose, 1× ASPA + N, 0.002 M MgSO₄, and 1× trace elements solution (4)). Conidia were inoculated into complete medium supplemented with 0.5 g/L casein peptone, 2.5 g/L yeast extract, 1% glucose, 1× ASPA + N, 0.002 M MgSO₄, and 1× trace elements solution (4) and incubated for 16 h at 30°C with shaking at 150 rpm. The mycelium was harvested, and protoplasts were prepared using 40 mg/mL VinoTaste (Novozymes, Bagsværd, Denmark) lysing enzymes in a buffer containing sorbitol, MES, and CaCl₂ (4), incubated at 37°C for 2.5 h. Protoplasts were resuspended in Zymo DNA/RNA Shield (Zymo Research, Irvine, USA).

Protoplasts of Aspergillus niger ATCC 1015 were sent to Plasmidosaurus for DNA extraction and custom sequencing using Oxford Nanopore Technology (ONT) and Illumina platform. Genomic DNA was isolated using the Monarch Genomic DNA Purification Kit (New England Biolabs, USA) without modifications and without shearing or size selection. Nanopore libraries (v14 chemistry; tagmentation) were prepared using the SQK-RBK114.96 kit (Oxford Nanopore Technologies, UK) and sequenced on a PromethION 24 using two R10.4.1 flow cells. Basecalling was performed with Guppy v6.4.6 (Oxford Nanopore Technologies; super-accurate mode, Q ≥ 10), and adapters and barcodes were trimmed using MinKNOW (Oxford Nanopore Technologies), yielding 521,857 reads in total (243,482 reads, N50 9,597 bp; 278,375 reads, N50 9,419 bp). Illumina libraries were prepared using the Illumina DNA Prep kit (Illumina, USA) and sequenced on a NextSeq 2000 platform as paired-end reads (2 × 150 bp), generating 28.5 million read pairs. Reads were first decontaminated against the human reference genome GRCh38.p13 (5): ONT using minimap2 v2.30 (6) and Illumina using bwa v0.7.19 (7). Unmapped ONT reads and Illumina read pairs were then extracted with samtools v1.22.1 (8). Finally, ONT reads were deduplicated (seqkit v2.10.1 (9)), quality-filtered (filtlong v0.3.0 (https://github.com/rrwick/Filtlong); top 95%, ≥3 kb), and Illumina reads were adapter-trimmed and quality- and length-filtered with fastp v1.0.1 (10) (Q ≥ 20, ≥50 bp).

The hybrid genome of A. niger ATCC 1015 was assembled de novo with Flye v2.9.6 in the nano-hq mode with an estimated genome size of 36 Mb (scaffolded) using processed Nanopore reads and polished with Illumina reads via Pilon v1.24 (11). Assembly quality was assessed using QUAST v5.3.0 (12) and BUSCO v5.7.1 (13) with the ascomycota_odb10 lineage data set, showing 99.6% BUSCO completeness. The assembly consists of nine contigs (including the mt-genome), with the nuclear genome composed of eight chromosomes at 35,617,925 bp (N50 4,304,314 bp; GC 49.59%) and one mtDNA at 31,333 bp. All chromosome-scale contigs terminate in tandem arrays with the motif TTTTAGGG, an elongated variant of the canonical telomeric repeat TTAGGG, which is also found in algae such as Chlamydomonas reinhardtii (14). Sequencing coverage was 59× for Nanopore reads and 197× for Illumina reads.

The mitochondrial contig was annotated using a reference-guided, manually curated approach, combining exonerate v2.4.0 (protein2genome model against Aspergillus flavus proteins [15]), tRNAscan-SE v2.0.12 (16) for tRNAs, and Infernal cmscan (Rfam) v1.1.5 for rRNAs; features were merged using AGAT v1.5.1 (17). The nuclear genome was annotated using funannotate v1.8.17 (18), including repeat masking, gene prediction with Augustus (19) (Aspergillus nidulans model), and functional annotation assigned by eggNOG-mapper v2.1.13 (20), resulting in 11,387 protein-coding genes. Liftoff v1.6.3 (21) was used to transfer features from the Aspni5 assembly (2), and the annotations were then merged using gffcompare v0.12.10 (22). During this process, protein ID-derived aliases were added, and hypothetical mRNAs were replaced with more informative gene names in the final GFF3 file.

DATA AVAILABILITY

Raw sequencing reads have been deposited in the Sequence Read Archive (SRA) under BioProject PRJNA1401865. Illumina short-read data are available under SRA run accession number SRR36864153. ONT long-read data are available under SRA run accession number SRR36864154. The nuclear whole-genome shotgun (WGS) project for Aspergillus niger ATCC 1015, associated with BioSample SAMN54575164, has been deposited in DDBJ/ENA/GenBank under the accession number JBTSWI000000000. The version described in this paper is the first version, JBTSWI010000000. The mitochondrial genome sequence has been deposited in DDBJ/ENA/GenBank under the accession number PX959665.