Corynebacterium mastitidis RC Genome Assembly Workflow

General scripts/code/pipeline/documentation used for assembling the Corynebacterium mastitidis RC genome sequence.

Authors: Vijay Nagarajan PhD, Amy Zhang PhD
Affiliation: Laboratory of Immunology, NEI/NIH
Contact: [email protected]
Description: This workflow takes the raw fastq files, checks the quality, does preprocessing, generates swarm command files for assembly and quality assessments
Platform: The workflow BASH commands were developed to run in the NIH Biowulf cluster computing facility, but could be reused/reproduced in any linux environment with appropriate changes
Publication: Nagarajan V, St Leger AJ, Zhang A, Silver P, Caspi RR. Draft Reference Genome Sequence of Corynebacterium mastitidis RC, an Ocular Commensal, Isolated from Mouse Conjunctiva. Microbiol Resour Announc. 2022 Jun 16;11(6):e0018722. doi: 10.1128/mra.00187-22. Epub 2022 May 10. PMID: 35536012; PMCID: PMC9202379.

1. Project Folder Structure

  cmast
    Tools
    RawData
    Results
      FastQC
      BBDUK
      UniCycler
      Abyss
      Quast
      CheckM
    Archive

2. Sequence Quality Analysis

# Set project path
projectpath="/data/cmast"
cd $projectpath

# FastQC
module load fastqc
module load multiqc
cd Results/FastQC

# Run fastqc quality analysis on raw data
find -L ../../RawData -name "*.fastq.gz" | parallel fastqc -o . {}

# Summarise fastqc reports
multiqc *fastqc.zip -n MultiFastQCReportRaw

3. Sequence Preprocessing

# BBTOOLS
module load bbtools
module load multiqc

# Identify adapters from list
bbtools bbduk in1=${projectpath}/RawData/1_S1_L001_R1_001.fastq.gz in2=${projectpath}/RawData/1_S1_L001_R2_001.fastq.gz k=23 ref=${projectpath}/RawData/adapters.fasta stats=${projectpath}/Results/BBDUK/stats.txt

# Remove adapters
cd ../../RawData
for i in {1..10}
	do
		bbtools bbduk in1=${i}_S${i}_L001_R1_001.fastq.gz in2=${i}_S${i}_L001_R2_001.fastq.gz out1=${projectpath}/Results/bbduk/${i}_S${i}_R1_trimmed.fastq.gz out2=${projectpath}/Results/bbduk/${i}_S${i}_R2_trimmed.fastq.gz ktrim=r k=21 mink=11 hdist=1 ref=${projectpath}/RawData/adapters.fasta 2> ${projectpath}/Results/bbduk/${i}_S${i}_log.txt
	done

# Run fastqc quality analysis on adapter cleaned data
find -L ${projectpath}/Results/bbduk -name "*.fastq.gz" | parallel fastqc -o . {}

# Summarise fastqc reports
multiqc FastQC/*fastqc.zip -n MultiFastQCReportRaw

4. Sequence Assembly

# Unicycler
# Assemble individual samples
# Prepare folders for bold/normal/conservative options
for i in {1..10}; do mkdir ${projectpath}/Results/unicycler/bold/${i}; done
for i in {1..10}; do mkdir ${projectpath}/Results/unicycler/normal/${i}; done
for i in {1..10}; do mkdir ${projectpath}/Results/unicycler/conservative/${i}; done

# Prepare swarm commands
for i in {1..10}; do echo "cd /data/cmast/Results/bbduk ; unicycler --depth_filter 0.00 -t 16 --mode bold -1 ${i}_S${i}_R1_trimmed.fastq.gz -2 ${i}_S${i}_R2_trimmed.fastq.gz -o /data/cmast/Results/unicycler/bold/${i}"; done
for i in {1..10}; do echo "cd /data/cmast/Results/bbduk ; unicycler --depth_filter 0.00 -t 16 --mode normal -1 ${i}_S${i}_R1_trimmed.fastq.gz -2 ${i}_S${i}_R2_trimmed.fastq.gz -o /data/cmast/Results/unicycler/normal/${i}"; done
for i in {1..10}; do echo "cd /data/cmast/Results/bbduk ; unicycler --depth_filter 0.00 -t 16 --mode conservative -1 ${i}_S${i}_R1_trimmed.fastq.gz -2 ${i}_S${i}_R2_trimmed.fastq.gz -o /data/cmast/Results/unicycler/conservative/${i}"; done

# Run swarm file
swarm -f unicycler_nofilter_adapter_removed_individual.swarm -g 64 -t 32 --gres=lscratch:200 --module unicycler

5. Assembly Quality Analysis

# Quast
# quast only
for i in {1..10}; do cp ${projectpath}/Results/unicycler/bold/${i}/assembly.fasta ${projectpath}/Results/unicycler/bold/${i}/${i}_b_assembly.fasta; quast.py -o ${projectpath}/Results/quast/alone/bold/${i} ${projectpath}/Results/unicycler/bold/${i}/${i}_b_assembly.fasta; done
for i in {1..10}; do cp ${projectpath}/Results/unicycler/normal/${i}/assembly.fasta ${projectpath}/Results/unicycler/normal/${i}/${i}_n_assembly.fasta; quast.py -o ${projectpath}/Results/quast/alone/normal/${i} ${projectpath}/Results/unicycler/normal/${i}/${i}_n_assembly.fasta; done
for i in {1..10}; do cp ${projectpath}/Results/unicycler/conservative/${i}/assembly.fasta ${projectpath}/Results/unicycler/conservative/${i}/${i}_c_assembly.fasta; quast.py -o ${projectpath}/Results/quast/alone/conservative/${i} ${projectpath}/Results/unicycler/conservative/${i}/${i}_c_assembly.fasta; done

# quast with reference 1
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/dsm/bold/${i} -r ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/bold/${i}/${i}_b_assembly.fasta; done
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/dsm/normal/${i} -r ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/normal/${i}/${i}_n_assembly.fasta; done
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/dsm/conservative/${i} -r ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/conservative/${i}/${i}_c_assembly.fasta; done

# quast with reference 2
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/16/bold/${i} -r ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/bold/${i}/${i}_b_assembly.fasta; done
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/16/normal/${i} -r ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/normal/${i}/${i}_n_assembly.fasta; done
for i in {1..10}; do quast.py -o ${projectpath}/Results/quast/16/conservative/${i} -r ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ${projectpath}/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/conservative/${i}/${i}_c_assembly.fasta; done

# Merge all transposted tsv quast reports into one tsv table
head -n 1 ${projectpath}/Results/quast/alone/bold/2/transposed_report.tsv > ${projectpath}/Results/quast/quast_merged_bbduk_alone.tsv
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/alone/bold/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_alone.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/alone/conservative/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_alone.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/alone/normal/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_alone.tsv ; done
head -n 1 ${projectpath}/Results/quast/dsm/bold/2/transposed_report.tsv > ${projectpath}/Results/quast/quast_merged_bbduk_dsm.tsv
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/dsm/bold/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_dsm.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/dsm/conservative/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_dsm.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/dsm/normal/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_dsm.tsv ; done
head -n 1 ${projectpath}/Results/quast/16/bold/2/transposed_report.tsv > ${projectpath}/Results/quast/quast_merged_bbduk_16.tsv
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/16/bold/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_16.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/16/conservative/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_16.tsv ; done
for i in {1..10}; do tail -n +2 ${projectpath}/Results/quast/16/normal/${i}/transposed_report.tsv >> ${projectpath}/Results/quast/quast_merged_bbduk_16.tsv ; done

# quast for four samples 1-4 combined assembly
quast.py -o ${projectpath}/Results/quast/1234/conservative ${projectpath}/Results/unicycler/conservative/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/normal ${projectpath}/Results/unicycler/normal/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/bold ${projectpath}/Results/unicycler/bold/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/dsm/conservative -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/conservative/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/dsm/normal -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/normal/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/dsm/bold -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/CmastDSM44356_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/bold/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/16/conservative -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/conservative/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/16/normal -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/normal/1234/assembly.fasta
quast.py -o ${projectpath}/Results/quast/1234/16/bold -r ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.fna.gz -g ../../../RCSM01_20170523-39981943/cmast-quast-Amy/NCBI_Cmast_Ref/Cmast16-1433_Ref_Genome.gff.gz ${projectpath}/Results/unicycler/bold/1234/assembly.fasta

# CheckM
# Assembly quality analysis
# use checkm.sh for individual builds
# checkm for 1234 combined assembly
cp ${projectpath}/Results/unicycler/bold/1234/assembly.fasta ${projectpath}/Results/unicycler/bold/1234/assembly.fna
cp ${projectpath}/Results/unicycler/conservative/1234/assembly.fasta ${projectpath}/Results/unicycler/conservative/1234/assembly.fna
cp ${projectpath}/Results/unicycler/normal/1234/assembly.fasta ${projectpath}/Results/unicycler/normal/1234/assembly.fna
checkm taxonomy_wf genus Corynebacterium ${projectpath}/Results/unicycler/bold/1234 ${projectpath}/Results/checkm/1234b-taxonomic/ -t 50
checkm taxonomy_wf genus Corynebacterium ${projectpath}/Results/unicycler/conservative/1234 ${projectpath}/Results/checkm/1234c-taxonomic/ -t 50

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Corynebacterium mastitidis RC Genome Assembly Workflow

1. Project Folder Structure

2. Sequence Quality Analysis

3. Sequence Preprocessing

4. Sequence Assembly

5. Assembly Quality Analysis

6. Swarm job files

7. BASH scripts

8. References

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Corynebacterium mastitidis RC Genome Assembly Workflow

1. Project Folder Structure

2. Sequence Quality Analysis

3. Sequence Preprocessing

4. Sequence Assembly

5. Assembly Quality Analysis

6. Swarm job files

7. BASH scripts

8. References

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