Metagenomics Annotation - P.generosa Water Samples Using BLASTn on Mox and KronaTools Visualization to Compare pH Treatments

Nearing the end of this quick metagenomics comparison of taxonomic differences between the two pH treatments (pH=7.1 and pH=8.2). Previously ran:

Here’s how the sample names breakdown:

Sample Develomental Stage (days post-fertilization) pH Treatment
MG1 13 8.2
MG2 17 8.2
MG3 6 7.1
MG5 10 8.2
MG6 13 7.1
MG7 17 7.1

After this completes, I’ll run KronaTools to get a rundown on taxonomic makeup of these two different pH treatments. I don’t expect BLASTn to take terribly long (based on previous metagenomics runs wit this data set); I’d guess around 6hrs.

SBATCH script (GitHub):

#!/bin/bash
## Job Name
#SBATCH --job-name=blastn_metagenomics
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=25-00:00:00
## Memory per node
#SBATCH --mem=120G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --workdir=/gscratch/scrubbed/samwhite/outputs/20190416_metagenomics_pgen_blastn

# Load Python Mox module for Python module availability

module load intel-python3_2017

# Document programs in PATH (primarily for program version ID)

date >> system_path.log
echo "" >> system_path.log
echo "System PATH for $SLURM_JOB_ID" >> system_path.log
echo "" >> system_path.log
printf "%0.s-" {1..10} >> system_path.log
echo ${PATH} | tr : \\n >> system_path.log


wd="$(pwd)"
threads=28

# Paths to programs
blast_dir="/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin"
blastn="${blast_dir}/blastn"

# Paths to blastdbs
blastdb_dir="/gscratch/srlab/blastdbs/ncbi-nr-nt-v5"
blast_db="${blastdb_dir}/nt"

# Directory with metagenemark FastAs
fasta_dir="/gscratch/scrubbed/samwhite/outputs/20190416_metagenomics_pgen_metagenemark"


# Export BLAST database directory
export BLASTDB=${blastdb_dir}

# Loop through metagenemark nucleotide FastAs
# Create list of those FastAs for reference
# Parse out sample names
# Run BLASTn on each FastA
for fasta in ${fasta_dir}/*nucleotides.fasta
do
  echo ${fasta} >> input.fasta.list.txt
  no_ext=${fasta%%.*}
  sample_name=$(echo ${no_ext##*/})
  # Run blastx on Trinity fasta
  ${blastn} \
  -query ${fasta} \
  -db ${blast_db} \
  -max_target_seqs 1 \
  -outfmt "6 std staxids" \
  -evalue 1e-10 \
  -num_threads ${threads} \
  > ${wd}/${sample_name}.blastn.outfmt6
done

RESULTS

Runtime was ~9hrs for these.

Output folder:

pH=7.1 BLASTn Output:

pH=8.2 BLASTn Output:

Interactive taxonomic Krona Plot (HTML):

Here are just a couple snapshots of the plots to add some “spice” to this notebook entry:


pH=7.1 Bacteria

pH=7.1 bacteria krona plot


pH=8.2 Bacteria

pH=8.2 bacteria krona plot


pH=7.1 Intramacronucleata (ciliate subphylum)

pH=7.1 Intramacronucleata krona plot


pH=8.2 Intramacronucleata (ciliate subphylum)

pH=8.2 Intramacronucleata krona plot