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Long read de novo Assembly exercise

Dataset is a HiFi Pacbio reads from the yeast Saccharomyces cerevisiae. The data can be downloaded from NCBI Sequence Read Archive (SRA)

  1. Download the reads for the following page or use sra-tools: SRR13577846
  • fastq-dump 3.0.10
  1. Quality check before assembly
  • FastQC 0.12.1
  1. de novo assembly
  • hifiasm 0.19.8-r603
  1. Quast quality assessment
  • QUAST 5.2.0
  1. BUSCO assessment
  • BUSCO 5.6.1

Setting up data

mkdir de_novo_genome_assembly

# Download the data from SRA 
fastq-dump --origfmt --split-files SRR13577846

Quality Control

mkdir fastQC_raw
fastqc --threads 6 SRR13577846_1.fastq --outdir fastQC_raw # --threads 6 to use 6 cores, --outdir to specify the output directory

De novo Assembly

mkdir hifiasm_1
hifiasm -o SRR13577846_1.asm -t32 -l0 /home/inf-51-2023/de_novo_genome_assembly/SRR13577846_1.fastq 2> SRR13577846_1.asm.log 
# -o to specify the output file, -t to specify the number of threads, -l to specify the minimum length of the reads

Change the gfa format to fasta

see this link (https://hifiasm.readthedocs.io/en/latest/faq.html)

awk '/^S/{print ">"$2;print $3}' SRR13577846_1.asm.bp.a_ctg.gfa > SRR13577846_1.asm.bp.a_ctg.fa

Do it for all the other gfa files

Assembly Quality Check with Quast

mkdir quast_analysis
# source of the reference genome, see this link https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000146045.2/

quast /home/inf-51-2023/de_novo_genome_assembly/hifiasm_1/SRR13577846_1.asm.bp.a_ctg.fa \
           /home/inf-51-2023/de_novo_genome_assembly/hifiasm_1/SRR13577846_1.asm.bp.p_ctg.fa \
           /home/inf-51-2023/de_novo_genome_assembly/hifiasm_1/SRR13577846_1.asm.bp.p_utg.fa \
           /home/inf-51-2023/de_novo_genome_assembly/hifiasm_1/SRR13577846_1.asm.bp.r_utg.fa \
        -r /home/inf-51-2023/de_novo_genome_assembly/ncbi_dataset/data/GCF_000146045.2/GCF_000146045.2_R64_genomic.fna \
        -o quast_analysis

Assembly Quality Check with Busco

fungi_odb10

busco -i hifiasm_1/SRR13577846_1.asm.bp.p_ctg.fa -l fungi_odb10 -m genome -c 24 -o fungi_out

saccharomycetes_odb10

busco -i hifiasm_1/SRR13577846_1.asm.bp.p_ctg.fa -l saccharomycetes_odb10 -m genome -c 24 -o