December 4, 2021 [metagenome] - sorting out the progress of metagenome process construction

• analytical framework
  •  

   

• Quality control
  • Filter
    • trimmomatic PE {input.R1} {input.R2} {output.R1_PE} {output.R1_UNPE} {output.R2_PE} {output.R2_UNPE} LEADING:3 TRAILING:3 SLIDINGWINDOW:5:20 MINLEN:50 -phred33
  • duplicate removal
    • echo \"{input.R1}\" > {params.sample_ID}_merge.fastuniq ;echo \"{input.R2}\" >> {params.sample_ID}_merge.fastuniq;fastuniq -i {params.sample_ID}_merge.fastuniq -t q -o {output.R1_uniq} -p {output.R2_uniq} -c 0
  • De hosting
    • bwa mem -k 30 -R \'@RG\\tID:foo\\tSM:bar\\tLB:Abace\' -t {threads} {params.genome} {input.R1} {input.R2} > {output}
    • samtools view -bS {input} -o {output}
    • samtools sort {input} -o {output}
    • samtools view {input.bam_file}|awk -F '\t' '$3==\"*\"{ {print $1}}'|uniq|seqtk subseq {input.R1} -> {output.nohost_file_R1};samtools view {input.bam_file}|awk -F '\t' '$3==\"*\"{ {print $1}}'|uniq|seqtk subseq {input.R2} -> {output.nohost_file_R2}
  • The report
    • gzip -c {input.nohost_file_R1} > {output.clean_R1}; gzip -c {input.nohost_file_R2} > {output.clean_R2};fastqc -o {output.fastqc_dir} --extract -q {output.clean_R1} {output.clean_R2}
• Reads-based
  • Species notes ：MetaPhIAn
    • cat {input.clean_R1} {input.clean_R2} > {params.combine_file};humann --input {params.combine_file} --output {output.result}
  • Function notes ：Humann
    • humann --input 02.align/nohost/4a_combine.fq --output 05.Annotation/4a/
  • Species notes ：Kraken2
• contigs-based
  • assemble ：Megahit
    • megahit -t {threads} -1 {input.clean_R1} -2 {input.clean_R2} -o {output.dir} --k-min 35 --k-max 95 --k-step 20 --min-contig-len 500 -m 0.1;cp {output.dir}/final.contigs.fa {output.assembly_fa}
  • Statistics Coverage：pileup
    • pileup.sh in={input.bam} ref={input.genome} out={output.covstats} overwrite=true
  • forecast ：prodigal
    • prodigal -i {input.contig} -o {output.gff} -f gff -p meta
  • Special notes
    • cd {params.sample_id};mkdir -p card dbCAN phi vfdb tcdb signalp;cd phi;/home/tanchaojun/anaconda3/envs/wgs/bin/diamond blastx -p 6 -k 1 -e 0.00001 --db /home/tanchaojun/database/phi/phi --query ../../../{input.genomic_cds} --out phi_result;cd ../vfdb;/home/tanchaojun/anaconda3/envs/wgs/bin/diamond blastx -p 6 -k 1 -e 0.00001 --db /home/tanchaojun/database/vfdb/vfdb_setA --query ../../../{input.genomic_cds} --out vfdb_result;cd ../tcdb;/home/tanchaojun/anaconda3/envs/wgs/bin/diamond blastx -p 6 -k 1 -e 0.00001 --db /home/tanchaojun/database/tcdb/tcdb --query ../../../{input.genomic_cds} --out tcdb_result;cd ../signalp;signalp -fasta ../../../{input.genomic_cds} -gff3 -mature -prefix signalp_result;cd ../dbCAN;/home/tanchaojun/anaconda3/envs/run_dbcan/bin/run_dbcan.py --db_dir /home/tanchaojun/database/dbCAN --hmm_cov 0.35 --hmm_eval 1e-15 --hmm_cpu 8 --dia_eval 1e-102 --dia_cpu 8 --out_dir ./ --out_pre dbCAN_result ../../../{input.genomic_cds} prok;cd ../card;/home/tanchaojun/anaconda3/envs/rgi/bin/rgi main -n 8 --input_sequence ../../../{input.genomic_cds} --output_file card_result --clean;
  • EGGNOG、CAZy、COG notes
    • cp {input.cds_fa} {params.sample_id}/cds.fa;cd {params.sample_id};mkdir -p eggnog COG;cd eggnog;emapper.py --cpu 20 --itype CDS -i ../../../{input.cds_fa} -o out --override -m diamond --evalue 0.001 --score 60 --pident 40 --query_cover 20 --subject_cover 20 --tax_scope auto --target_orthologs all --go_evidence non-electronic --pfam_realign none --report_orthologs --decorate_gff yes --data_dir /home/tanchaojun/anaconda3/envs/eggnog/lib/python3.7/site-packages/data;cd ../../../
    • cp scripts/anno/COG/* {params.sample_id};cp scripts/anno/eggnog/* {params.sample_eggnog};cd {params.sample_eggnog};perl emapper2anno.pl out.emapper.annotations > ../COG/eggnog.anno.xls;cd ../COG;python COG.py fun2003-2014.tab ../eggnog/out.emapper.annotations;/usr/bin/Rscript 7.eggnog.plot.R DrawAnnotationPic.R.txt COG.pdf
    • mkdir -p {params.CAZy_dir}&&cd {params.CAZy_dir};/home/tanchaojun/anaconda3/envs/run_dbcan/bin/run_dbcan.py --db_dir /home/tanchaojun/database/dbCAN --hmm_cov 0.35 --hmm_eval 1e-15 --hmm_cpu 8 --dia_eval 1e-102 --dia_cpu 8 --out_dir ./ --out_pre dbCAN_result ../../../{input.cds_fa} prok
  • Species notes ：NR（ Not yet in ）
• Post analysis ( Not completed yet )
  • Species and functional composition analysis
    • Venn
    • heatmap
    • species 、 Functional diagram
    • Species composition diagram
  • Sample comparison and analysis
    • UPGMA Clustering analysis
    • Hierarchical clustering heat map
    • PCA
    • PCoA
  • Comparison and analysis between groups
    • AMOVA