Metagenomic assembly pipeline

Assembly exercise workflow for Nordic Summer School on Computational Microbiome Research, 2024.

Data

Illumina, Nanopore and Pacbio RS II sequencing data from 12 species mock community: Shotgun metagenome data of a defined mock community using Oxford Nanopore, PacBio and Illumina technologies

Sequence data

Accession	Platform	Data in Gb
SRR8073715	Pacbio RS II	1.1
SRR8073714	PacBio RS II	1.5
SRR8073713	Nanopore	3.7
SRR8073716	Illumina	64

Data download

Download read files and pack them.

for acc in $(cat accessions.txt)
do	
  fasterq-dump -e $SLURM_CPUS_PER_TASK -O data --split-3 --skip-technical --progress $acc
  pigz ${acc}*.fastq
done

Subsample Illumina data

Keep 10 % of original reads (~6.4Gb).

seqkit sample -s 100 data/SRR8073716_1.fastq.gz  -p 0.1 |pigz -c > data/SRR8073716_sub_1.fastq.gz
seqkit sample -s 100 data/SRR8073716_2.fastq.gz  -p 0.1 |pigz -c > data/SRR8073716_sub_2.fastq.gz

Assemblies

Illumina only with spades

spades.py \
    --meta \
    -1 data/SRR8073716_sub_1.fastq.gz \
    -2 data/SRR8073716_sub_2.fastq.gz \
    --only-assembler \
    -o illumina_only

Pacbio only with metaflye (use both pacbio runs).

flye \
    --meta \
    --pacbio-raw data/SRR8073715.fastq.gz data/SRR8073714.fastq.gz \
    --out-dir pacbio_only

Nanopore only with metaflye.

flye \
    --meta \
    --nano-raw data/SRR8073713.fastq.gz \
    --out-dir nanopore_only

Illumina + Pacbio hybrid with spades

spades.py \
   --meta \
   -1 data/SRR8073716_sub_1.fastq.gz \
   -2 data/SRR8073716_sub_2.fastq.gz \
   --pacbio data/SRR8073715.fastq.gz \
   --pacbio data/SRR8073714.fastq.gz \
   --only-assembler \
   -o pacbio_hybrid

Illumina + Nanopore hybrid with spades.

spades.py \
   --meta \
   -1 data/SRR8073716_sub_1.fastq.gz \
   -2 data/SRR8073716_sub_2.fastq.gz \
   --nanopore data/SRR8073713.fastq.gz \
   --only-assembler \
   -o nanopore_hybrid

Download links for the assemblies

Illumina only: https://a3s.fi/antkark-2001183-pub/illumina_only.fasta
Nanopore only: https://a3s.fi/antkark-2001183-pub/nanopore_only.fasta
Pacbio only: https://a3s.fi/antkark-2001183-pub/pacbio_only.fasta
Nanopore hybrid: https://a3s.fi/antkark-2001183-pub/nanopore_hybrid.fasta
Pacbio hybrid: https://a3s.fi/antkark-2001183-pub/pacbio_hybrid.fasta

Assembly stats

Assembly statistics with metaquast for all assemblies.

metaquast.py \
   -o assembly_stats \
   --max-ref-number \
   0 */contigs.fasta */assembly.fasta

Software versions:

fasterq-dump v.3.0.0
seqkit v.0.16.0
spades v.4.0.0
flye v.2.9.4-b1799
metaquast v.5.2.0

karkman/MicrobiomeSummerSchool2024