Bioinformatic_pipeline

Taxonomy profile

Kraken

Make sure ~/kraken_db exists Clean read name as \${i}_1.fastq.gz & ${i}_2.fastq.gz
Put all clean read file in $~/clean_read
Create list in $~/sample_list/clean_read_list

Usage

~/bioinformatic_shell_script/kraken2.sh   

ARG profile

DeepARG

Clean read name as \${i}_1.fastq.gz & ${i}_2.fastq.gz
Put all clean read file in $~/clean_read
Create list in $~/sample_list/clean_read_list

Usage

conda activate deeparg_env    
~/bioinformatic_shell_script/deeparg.sh   
conda deactivate    

ARG-OAP

Clean read name as \${i}_1.fq.gz & ${i}_2.fq.gz
Create meta-data.txt in $~/sample_list

Usage

singularity exec -B ~/clean_read ~/arg-oap/argoapv2.5.sif /home/argsoapv2.5/argoap_version2.5 -i ~/clean_read -m ~/sample_list/meta-data.txt -o ~/arg-oap -n 16 -z  

Functional profile

HUMAnN 3.0

Make sure ~/humann_database exists Clean read name as \${i}_1.fastq.gz & ${i}_2.fastq.gz
Put all clean read file in $~/clean_read
Create list in $~/sample_list/clean_read_list

Usage

conda activate mpa    
~/bioinformatic_shell_script/humann.sh  
conda deactivate

Binning pipeline

Co-assembly (MEGAHIT)

Clean read name as \${i}_1.fastq.gz & ${i}_2.fastq.gz
Put all clean read file in $~/clean_read

#metawrap_coassembly
cat ~/clean_read/*_1.fastq.gz > ~/clean_read/all_reads_1.fastq.gz
cat ~/clean_read/*_2.fastq.gz > ~/clean_read/all_reads_2.fastq.gz  
{Then remove all individual clean read manually}
gunzip ~/clean_read/all_reads_1.fastq.gz ~/clean_read/all_reads_2.fastq.gz
megahit -t 16 -m 0.95 -1 ~/clean_read/all_reads_1.fastq -2 ~/clean_read/all_reads_2.fastq --min-contig-len 1000 -o ~/megahit/megehit_coassembly   
rm ~/clean_read/all_reads_1.fastq ~/clean_read/all_reads_2.fastq

megahit -t 16 -m 0.95 -1 AT1_1.fastq.gz,AT2_1.fastq.gz,AT3_1.fastq.gz,AT4_1.fastq.gz,AT5_1.fastq.gz,AP1_1.fastq.gz,AP2_1.fastq.gz,AP3_1.fastq.gz,AP4_1.fastq.gz,AP5_1.fastq.gz,OP1_1.fastq.gz,OP2_1.fastq.gz,OP3_1.fastq.gz,OP4_1.fastq.gz,OP5_1.fastq.gz -2 AT1_2.fastq.gz,AT2_2.fastq.gz,AT3_2.fastq.gz,AT4_2.fastq.gz,AT5_2.fastq.gz,AP1_2.fastq.gz,AP2_2.fastq.gz,AP3_2.fastq.gz,AP4_2.fastq.gz,AP5_2.fastq.gz,OP1_2.fastq.gz,OP2_2.fastq.gz,OP3_2.fastq.gz,OP4_2.fastq.gz,OP5_2.fastq.gz --min-contig-len 1000 -o ~/megahit/megehit_coassembly --presets meta-large

bbnorm.sh in1=OP5_1.fastq in2=OP5_2.fastq out1=nOP5_1.fastq out2=nOP5_2.fastq target=70

megahit -t 16 -m 0.95 -1 AT1_1.fastq,AT2_1.fastq,AT3_1.fastq,AT4_1.fastq,AT5_1.fastq,AP1_1.fastq,AP2_1.fastq,AP3_1.fastq,AP4_1.fastq,AP5_1.fastq,OP1_1.fastq,OP2_1.fastq,OP3_1.fastq,OP4_1.fastq,OP5_1.fastq -2 AT1_2.fastq,AT2_2.fastq,AT3_2.fastq,AT4_2.fastq,AT5_2.fastq,AP1_2.fastq,AP2_2.fastq,AP3_2.fastq,AP4_2.fastq,AP5_2.fastq,OP1_2.fastq,OP2_2.fastq,OP3_2.fastq,OP4_2.fastq,OP5_2.fastq --min-contig-len 1000 -o ~/megahit/megehit_coassembly

#quast
~/quast-5.1.0rc1/quast.py ~/megahit/megehit_coassembly/final.contigs.fa -o ~/megahit/megehit_coassembly/coassembly_quast

metaWRAP (use fastq format except metabat2)

metabat2

cd ~/bowtie2    
bowtie2-build  ~/megahit/megehit_coassembly/final.contigs.fa coassembly_contig   

copy clean reads to $bowtie2

bowtie2 -x coassembly_contig -1 {read1_1.fastq.gz,read2_1.fastq.gz,...} -2  {read1_2.fastq.gz,read2_2.fastq.gz,...} | samtools sort -o coassembly.sort.bam  

delete clean reads in $bowtie2

conda activate metawrap-env
cd ~/metabat2
runMetaBat.sh ~/megahit/megehit_coassembly/final.contigs.fa ~/bowtie2/coassembly.sort.bam   
mv {final.contigs.fa.metabat-bins-20220508_131755} metabat2_bins
rm ~/metabat2/final.contigs.fa.depth.txt  
mv ~/metabat2/metabat2_bins ~/metawrap_run/initial_binning

metaWRAP binning (maxbin2, concoct)

metawrap binning -o ~/metawrap_run/initial_binning -t 16 -a ~/megahit/megehit_coassembly/final.contigs.fa --maxbin2 --concoct ~/clean_read/*fastq    

metaWRAP refinement

metawrap bin_refinement -o ~/metawrap_run/bin_refinement -t 16 -A ~/metawrap_run/initial_binning/metabat2_bins -B ~/metawrap_run/initial_binning/maxbin2_bins -C  ~/metawrap_run/initial_binning/concoct_bins -c 50 -x 10 -m 56 

#If error, maybe re-run concoct binning
metawrap binning -o ~/metawrap_run/initial_binning -t 16 -a ~/megahit/megehit_coassembly/final.contigs.fa --concoct ~/clean_read/*fastq

metaWRAP quant_bins

metawrap quant_bins -b ~/metawrap_run/bin_refinement/metawrap_50_10_bins -o ~/metawrap_run/bin_quant -a ~/megahit/megehit_coassembly/final.contigs.fa ~/clean_read/*fastq -t 16  
conda deactivate

GTBDK

Usage

conda activate gtdbtk-1.5.0   
gtdbtk classify_wf --genome_dir ~/metawrap_run/bin_refinement/metawrap_50_10_bins -x fa --out_dir ~/bins_gtdbdk --scratch_dir scratch.tempory --cpus 16    
conda deactivate

Bin Prodigal & CD-HIT

Put all bin file in $~/metawrap_run/bin_refinement/metawrap_50_10_bins

Usage

#Edit shell script  
vim ~/bioinformatic_shell_script/bin_prodigal_cdhit.sh  
for i in {1..[number]}  

~/bioinformatic_shell_script/bin_prodigal_cdhit.sh  

DIAMOND-blastx (ARGs, MGEs) & abricate (VFs)

Usage

#Edit shell script  
vim ~/bioinformatic_shell_script/DIAMOND-blastx_abricate.sh 
for i in {1..[number]} 

~/bioinformatic_shell_script/DIAMOND-blastx_abricate.sh  

Seqkit_Bowtie2_BBmap (genes coverage quantification)

R script

make sure $//wsl$/Ubuntu/home/yen/ARG_MGE_manually_gtf/ exist
run DIAMOND_ARG_MGE_VF.R
remove 0kb file in $//wsl$/Ubuntu/home/yen/ARG_MGE_manually_gtf/

shell script

Create $~/sample_list/sample_type_list

Usage

#change for loop i to bin numbers
vim ~/bioinformatic_shell_script/ARG_MGE_coverage.sh  
  
#run
~/bioinformatic_shell_script/ARG_MGE_coverage.sh  

ARG_Ranker

Merge forward & reverse read

Clean read name as \${i}_1.fastq & ${i}_2.fastq
Put all clean read file in $~/clean_read
Create list in $~/sample_list/clean_read_list

Usage

~/bioinformatic_shell_script/merge_read12.sh  

Run

Usage

mkdir arg_ranker_output
arg_ranker -i ~/clean_read -kkdb ~/kraken_db -t 16 -o ~/arg_ranker_output   
sh /home/yen/arg_ranker_output/script_output//arg_ranker.sh   

ARG chromosome & plasmid distribution

Assembly, Prodigal & ARG blastx

Individual read assembly

Clean read name as \${i}_1.fastq.gz & ${i}_2.fastq.gz
Put all clean read file in $~/clean_read
Create list in $~/sample_list/clean_read_list

Usage

~/bioinformatic_shell_script/megahit_individual.sh  

Prodigal & ARG blastx

Usage

~/bioinformatic_shell_script/assembly_arg_diamond.sh  

R script

make sure $//wsl$/Ubuntu/home/yen/individual_assembly_arg_manually_gff/ exist
run ARG_individual_assembly.R

Extract >1000bp contigs

Usage

~/bioinformatic_shell_script/length_1000bp.sh  

PlasFlow

Usage

conda activate plasflow
~/bioinformatic_shell_script/plasflow.sh
conda deactivate

MetaCompare

Create list in $~/sample_list/clean_read_list
Make sure ~/megahit/megehit_individual/${i}_assembly/final.contigs.fa & ~/assembly_prodigal/${i}_assembly.nucl exist

Usage

~/bioinformatic_shell_script/metacompare.sh
#This script will rename final.contigs.fa to ${i}_final.contigs.fa as well

Sourcetracker2

Prepare metatest.txt in ~/sourcetracker2
Change file name: ~/kraken2_bracken/${i}_kraken2.report to ~/kraken2_bracken/${i}.report

Usage

cd ~/sourcetracker2
kraken-biom  ~/kraken2_bracken/*kraken2.report --max G --fmt tsv
kraken-biom  ~/kraken2_bracken/*kraken2.report --max G
conda activate st2
sourcetracker2 gibbs -i ~/sourcetracker2/table.biom -m ~/sourcetracker2/metatest.txt -o ~/sourcetracker2/sourcetracker2.output

DIAMOND-blastx (SARGs, MGEs , VFs)

Usage

#Edit shell script  
vim ~/bioinformatic_shell_script/SARG_MGEs_VFs_MRGs_diamond.sh 
for i in {1..[number]} 

~/bioinformatic_shell_script/SARG_MGEs_VFs_MRGs_diamond.sh

Seqkit_Bowtie2_BBmap (genes coverage quantification)

R script

make sure $//wsl$/Ubuntu/home/yen/manual_gtf_2.0/ exist
run diamond_SARG_MGE_VF.R
remove 0kb file in $//wsl$/Ubuntu/home/yen/ARG_MGE_manually_gtf/

shell script

Create $~/sample_list/sample_type_list

Usage

#change for loop i to bin numbers
vim ~/bioinformatic_shell_script/SARG_MGE_MRG_coverage.sh 
  
#run
~/bioinformatic_shell_script/SARG_MGE_MRG_coverage.sh

Binning_Plasflow

Usage

conda activate plasflow
mkdir ~/Binning_PlasFlow
PlasFlow.py --input ~/megahit/megehit_coassembly/final.contigs.fa --output ~/Binning_PlasFlow/coassembly.plasflow_predictions.tsv --threshold 0.7
conda deactivate

Individual assembly (diamond_SARG_MGE_MRG)

Usage

#CD-HIT & diamond
~/bioinformatic_shell_script/individual_assembly_diamond.sh

Individual_assembly_plasflow

Usage

conda activate plasflow
~/bioinformatic_shell_script/indiv_assembly_Plasflow.sh
conda deactivate

contigs_taxa_kraken2

Usage

~/bioinformatic_shell_script/contigs_kraken2.sh

ARG_contigs_coverage

Usage

~/bioinformatic_shell_script/coverage_indi_ARG_contigs.sh

ARG_contigs_ORF

Usage

~/bioinformatic_shell_script/coverage_indi_ARG_ORF.sh

MGE_contigs_ORF

Usage

~/bioinformatic_shell_script/coverage_indi_MGE_ORF.sh