As part of our Panopea generosa (Pacific geoduck) genome sequencing efforts, Steven came across a tool designed to help identify if there are any contaminating sequences in your assembly. The software is BlobToolKit. The software is actually a complex pipeline of separate tools ([minimap2])https://github.com/lh3/minimap2, BLAST, DIAMOND BLAST, and BUSCO) which aligns sequencing reads and assigns taxonomy to the reads, as well as marking regions of the assembly with various taxonomic assignments.

The pipeline has been regularly updated and when I initially tried to run it, failed because the pipeline required an internet connection. Side note, the tool was really written to analyze all of the genome assemblies that exist in NCBI. As such, it was written to automatically download assemblies/reads while running things. Considering the amount of data I had, this could only be run on Mox, which does not have internet access for any submitted jobs. The developer has been remarkably helpful and worked to update the pipeline to remove this barrier, allowing for local (i.e. offline) analysis. There were some other bumps along the way and the developer addressed those issues, too! One great aspect of this pipeline is that it’s written as a Snakemake pipeline, which incorporates checkpoints. This allowed me to continue the pipeline each time it died due to some weird bug here and there. This job would’ve taken much longer if I had to restart it from the beginning each time.

The job was run on Mox.

SBATCH script (GitHub):

20210712_pgen_blobtools_Panopea-generosa-v1.0.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=20210712_pgen_blobtools_Panopea-generosa-v1.0
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=10-00:00:00
## Memory per node
#SBATCH --mem=200G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210712_pgen_blobtools_Panopea-generosa-v1.0


### Script to run the Blobtools2 Pipeline
### on trimmed 10x Genomics/HiC FastQs from 20210401.
### Using to identify sequencing contaminants in Panopea-generosa-v1.0 genome assembly
### Generates a Snakemake config file
### Outputs Blobtools2 JSON files for use in the Blobtools2 viewer

### Utilizes NCBI taxonomy dump and customized UniProt database for DIAMOND BLASTx

### Requires Anaconda to be in system $PATH!

### Follows instructions for release v2.6.0 (https://github.com/blobtoolkit/pipeline/tree/release/v2.6.0)
### And changes noted in this GitHub Issue: https://github.com/blobtoolkit/pipeline/issues/13#issuecomment-868510883

### MODIFED HARD-CODED THREAD COUNTS IN rules/run_minimap2_index.smk and rules/run_minimap2_align.smk

###################################################################################
# These variables need to be set by user

# Load blobtoolkit module to add btk to PATH
module load blobtoolkit-v2.6.1.module

# Set working directory
wd=$(pwd)
echo "Working directory is ${wd}."
echo ""

# Set base directory for blobltools structure
base_dir=${wd}/blobtoolkit

# Pipeline options
## BLASTn evalue
evalue="1.0e-10"

## NCBI Tax ID and genus/species
ncbi_tax_id=1049056
species="Panopea generosa"

## NCBI Taxonomy Root ID to begin from
root=1

# Set number of CPUs to use
threads=40

# Input/output files
assembly_name=Panopea_generosa_v1
orig_fasta=/gscratch/srlab/sam/data/P_generosa/genomes/Panopea-generosa-v1.0.fa
fastq_checksums=fastq_checksums.md5
trimmed_reads_dir=/gscratch/scrubbed/samwhite/outputs/20210401_pgen_fastp_10x-genomics

## New genome name for BTK filename requirements (no periods)
genome_fasta=${wd}/Panopea_generosa_v1.fasta.gz

# Programs
## Blobtools2 directory
blobtools2=/gscratch/srlab/programs/blobtoolkit-v2.6.1/blobtools2

## BTK pipeline directory
btk_pipeline=/gscratch/srlab/programs/blobtoolkit-v2.6.1/pipeline

## Conda environment directory
conda_dir=/gscratch/srlab/programs/anaconda3/envs/btk_env


# Databases
## BUSCO lineage database directory
busco_dbs=/gscratch/srlab/sam/data/databases/BUSCO

## Blobtools NCBI taxonomy database directory
btk_ncbi_tax_dir=/gscratch/srlab/blastdbs/20210401_ncbi_taxonomy

## NCBI nt database dir
ncbi_db=/gscratch/srlab/blastdbs/20210401_ncbi_nt
ncbi_db_name="nt"

## Uniprot DIAMOND database dir
uniprot_db=/gscratch/srlab/blastdbs/20210401_uniprot_btk
uniprot_db_name=reference_proteomes

# Programs associative array
declare -A programs_array
programs_array=()


###################################################################################

# Exit script if any command fails
set -e

# Load Anaconda
# Uknown why this is needed, but Anaconda will not run if this line is not included.
. "/gscratch/srlab/programs/anaconda3/etc/profile.d/conda.sh"

# Gzip FastA - needed for blobltoolkit to run properly
if [ ! -f "${genome_fasta}" ]; then
  gzip -c ${orig_fasta} > "${genome_fasta}"
fi

# Generate checksum for "new" FastA
if [ ! -f "${genome_fasta}" ]; then
  md5sum "${genome_fasta}" > genome_fasta.md5
fi

# Concatenate all R1 reads
if [ ! -f "reads_1.fastq.gz" ]; then
  for fastq in "${trimmed_reads_dir}"/*R1*.fq.gz
  do
    echo ""
    echo "Generating checksum for ${fastq}"
    md5sum "${fastq}" >> ${fastq_checksums}
    echo "Checksum generated for ${fastq}."

    echo ""
    echo "Concatenating ${fastq} to reads_1.fastq.gz"
    cat "${fastq}" >> reads_1.fastq.gz
    echo "Finished concatenating ${fastq} to reads_1.fastq.gz"
  done
fi

# Concatenate all R2 reads
if [ ! -f "reads_2.fastq.gz" ]; then
  for fastq in "${trimmed_reads_dir}"/*R2*.fq.gz
  do
    echo ""
    echo "Generating checksum for ${fastq}"
    md5sum "${fastq}" >> ${fastq_checksums}
    echo "Checksum generated for ${fastq}."

    echo ""
    echo "Concatenating ${fastq} to reads_2.fastq.gz"
    cat "${fastq}" >> reads_2.fastq.gz
    echo "Finished concatenating ${fastq} to reads_2.fastq.gz"
  done
fi

# Count scaffolds in assembly
scaffold_count=$(grep -c ">" "${genome_fasta}")

# Count nucleotides in assembly
genome_nucleotides_count=$(grep -v ">" "${genome_fasta}" | wc | awk '{print $3-$1}')

# Create BTK config YAML
# BUSCO lineage order is important! List in order of most taxonomic specific to most general.
if [ -f "config.yaml" ]; then
  rm "config.yaml"
fi
{
  printf "%s\n" "assembly:"
  printf "%2s%s\n" "" "accession: draft" "" "file: ${genome_fasta}" "" "level: scaffold" "" "scaffold-count: ${scaffold_count}" "" "span: ${genome_nucleotides_count}"
  printf "%2s%s\n" "" "prefix: ${assembly_name}"
  printf "%s\n" "busco:"
  printf "%2s%s\n" "" "download_dir: ${busco_dbs}"
  printf "%2s%s\n" "" "lineages:"
  printf "%4s%s\n" "" "- arthropoda_odb10" "" "- eukaryota_odb10" "" "- metazoa_odb10" "" "- bacteria_odb10" "" "- archaea_odb10"
  printf "%2s%s\n" "" "basal_lineages:"
  printf "%4s%s\n" "" "- archaea_odb10" "" "- bacteria_odb10" "" "- eukaryota_odb10"
  printf "%s\n" "reads:"
  printf "%2s%s\n" "" "paired:"
  printf "%4s%s\n" "" "- prefix: reads"
  printf "%6s%s\n" "" "platform: ILLUMINA" "" "file: ${wd}/reads_1.fastq.gz;${wd}/reads_2.fastq.gz"
  printf "%s\n" "settings:"
  printf "%2s%s\n" "" "taxdump: ${btk_ncbi_tax_dir}"
  printf "%2s%s\n" "" "blast_chunk: 100000"
  printf "%2s%s\n" "" "blast_max_chunks: 10"
  printf "%2s%s\n" "" "blast_overlap: 0"
  printf "%2s%s\n" "" "blast_min_length: 1000"
  printf "%s\n" "similarity:"
  printf "%2s%s\n" "" "defaults:"
  printf "%4s%s\n" "" "evalue: ${evalue}" "" "max_target_seqs: 10" "" "import_evalue: 1.0e-25" "" "taxrule: buscogenes"
  printf "%2s%s\n" "" "diamond_blastx:"
  printf "%4s%s\n" "" "name: ${uniprot_db_name}" "" "path: ${uniprot_db}"
  printf "%2s%s\n" "" "diamond_blastp:"
  printf "%4s%s\n" "" "name: ${uniprot_db_name}" "" "path: ${uniprot_db}" "" "import_max_target_seqs: 100000"
  printf "%2s%s\n" "" "blastn:"
  printf "%4s%s\n" "" "name: ${ncbi_db_name}" "" "path: ${ncbi_db}"
  printf "%s\n" "taxon:"
  printf "%2s%s\n" "" "name: ${species}" "" "taxid: '${ncbi_tax_id}'"
} >> config.yaml

# Activate blobtoolkit conda environment
conda activate btk_env

# Run snakemake, btk pipeline
snakemake -p \
--use-conda \
--conda-prefix ${conda_dir} \
--directory "${base_dir}" \
--configfile "${wd}"/config.yaml \
--stats ${assembly_name}.blobtoolkit.stats \
-j ${threads} \
--rerun-incomplete \
-s ${btk_pipeline}/blobtoolkit.smk \
--resources btk=1


###################################################################################

# Capture program options
if [[ "${#programs_array[@]}" -gt 0 ]]; then
  echo "Logging program options..."
  for program in "${!programs_array[@]}"
  do
    {
    echo "Program options for ${program}: "
    echo ""
    # Handle samtools help menus
    if [[ "${program}" == "samtools_index" ]] \
    || [[ "${program}" == "samtools_sort" ]] \
    || [[ "${program}" == "samtools_view" ]]
    then
      ${programs_array[$program]}

    # Handle DIAMOND BLAST menu
    elif [[ "${program}" == "diamond" ]]; then
      ${programs_array[$program]} help

    # Handle NCBI BLASTx menu
    elif [[ "${program}" == "blastx" ]]; then
      ${programs_array[$program]} -help
    fi
    ${programs_array[$program]} -h
    echo ""
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
  } &>> program_options.log || true

    # If MultiQC is in programs_array, copy the config file to this directory.
    if [[ "${program}" == "multiqc" ]]; then
      cp --preserve ~/.multiqc_config.yaml multiqc_config.yaml
    fi
  done
fi

# Document programs in PATH (primarily for program version ID)
{
  date
  echo ""
  echo "System PATH for $SLURM_JOB_ID"
  echo ""
  printf "%0.s-" {1..10}
  echo "${PATH}" | tr : \\n
} >> system_path.log

echo "Finished logging system PATH"

RESULTS

Usually, I post the run time for my Mox jobs here. However, this was stopped/started numerous times, making it difficult to actually determine the cumulative runtime.

Output folder:

20210712_pgen_blobtools_Panopea-generosa-v1.0/
- JSON output directory
  - 20210712_pgen_blobtools_Panopea-generosa-v1.0/blobtools/Panopea_generosa_v1/
  - These files are needed to use with the Blobtools viewer software.

Next up will be loading the JSON output files into the Blobtools viewer and analysing taxonomic assignments of reads/regions throughout our genome assembly.

RESULTS

JSON output directory