Methods

Data Acquisition and Quality Assessment

Raw sequencing data were obtained from the NCBI Sequence Read Archive (SRA). Two datasets were downloaded using prefetch from the SRA Toolkit:

  • Illumina paired-end reads: SRR21149832
  • PacBio HiFi long reads: SRR21149833
# Download SRA data
prefetch SRR21149832
prefetch SRR21149833

The SRA files were converted into FASTQ format using fasterq-dump. Illumina data were split into forward and reverse strands, while PacBio HiFi reads were output as a single file.

# Convert Illumina reads and split
fasterq-dump --split-files --skip-technical -M 30 SRR21149832

# Convert PacBio reads (single output file)
fasterq-dump --split-files --skip-technical -M 30 SRR21149833

Initial quality control was performed using FastQC on all FASTQ files to assess per-base sequence quality, GC content, and potential contamination.

Adapter Removal

For the Illumina reads, adapter sequences were trimmed using NGMerge, with parameters optimized for paired-end reads:

NGmerge -a -1 SRR21149832_1.fastq -2 SRR21149832_2.fastq -o outputfile -v -u 73 -n 1

PacBio HiFi reads were not adapter-trimmed as Hifiasm tolerates potential adapters during assembly.

Genome Assembly

Two separate genome assemblies were conducted:

Illumina short reads: assembled using SPAdes (v3.15.4)

PacBio HiFi long reads: assembled using Hifiasm (v0.16.1)

Job scripts were executed on a high-memory cluster with the following settings:

# SPAdes job script
spades.py --isolate -1 removed_adapt_1.fastq -2 removed_adapt_2.fastq -o ./illumina_results

# Hifiasm assembly
hifiasm -o pacb.asm -t 1 SRR21149833.fastq

The PacBio output .gfa was decompressed to .fa format:

gunzip pacb.asm.bp.p_ctg.gfa

Assembly Evaluation with QUAST and BUSCO

The QUAST tool was used to evaluate contiguity metrics (e.g. N50, total length, GC content) against the reference genome:

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/027/921/745/GCF_027921745.1_ASM2792174v1/GCF_027921745.1_ASM2792174v1_genomic.fna.gz

QUAST was then run using the reference and multiple assemblies:

quast.py -r GCF_027921745.1_ASM2792174v1_genomic.fna -o quast_compare contigs.fasta scaffolds.fasta pacb.asm.bp.p_ctg.fa

BUSCO (Benchmarking Universal Single-Copy Orthologs) was used for completeness assessment against the ascomycota_odb12 lineage:

# For PacBio
busco -m genome -i pacb.asm.bp.p_ctg.fa -o busco_pacbio -f -l ascomycota_odb12

# For Illumina (contigs and scaffolds) and reference
busco -m genome -i contigs.fasta -o busco_illumina_contigs -f -l ascomycota_odb12
busco -m genome -i scaffolds.fasta -o busco_illumina_scaffolds -f -l ascomycota_odb12
busco -m genome -i reference_genome.fa -o busco_reference -f -l ascomycota_odb12

Reference-Guided Scaffolding with RagTag

Both assemblies were scaffolded using RagTag, utilizing the downloaded reference genome to improve structural contiguity:

ragtag.py scaffold GCF_027921745.1_ASM2792174v1_genomic.fna pacb.asm.bp.p_ctg.fa -o ragtag_pacbio -t 1
ragtag.py scaffold GCF_027921745.1_ASM2792174v1_genomic.fna contigs.fasta -o ragtag_illumina -t 1

Final QUAST evaluations were also conducted on the RagTag-scaffolded outputs using the same command as above.