Interhost variant calling (Data_Tam_DNAseq_2026_19606wt_dAB_dIJ_mito_flu_on_ATCC19606)

Input data:

 mkdir bacto; cd bacto;
 mkdir raw_data; cd raw_data;

 # ── Fluoxetine Dataset ──
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcef-1/19606△ABfluEcef-1_1.fq.gz flu_dAB_cef_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcef-1/19606△ABfluEcef-1_2.fq.gz flu_dAB_cef_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcipro-2/19606△ABfluEcipro-2_1.fq.gz flu_dAB_cipro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcipro-2/19606△ABfluEcipro-2_2.fq.gz flu_dAB_cipro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEdori-2/19606△ABfluEdori-2_1.fq.gz flu_dAB_dori_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEdori-2/19606△ABfluEdori-2_2.fq.gz flu_dAB_dori_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEnitro-3/19606△ABfluEnitro-3_1.fq.gz flu_dAB_nitro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEnitro-3/19606△ABfluEnitro-3_2.fq.gz flu_dAB_nitro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEpip-1/19606△ABfluEpip-1_1.fq.gz flu_dAB_pip_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEpip-1/19606△ABfluEpip-1_2.fq.gz flu_dAB_pip_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEpolyB-3/19606△ABfluEpolyB-3_1.fq.gz flu_dAB_polyB_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEpolyB-3/19606△ABfluEpolyB-3_2.fq.gz flu_dAB_polyB_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEtet-1/19606△ABfluEtet-1_1.fq.gz flu_dAB_tet_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEtet-1/19606△ABfluEtet-1_2.fq.gz flu_dAB_tet_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEcef-4/19606△IJfluEcef-4_1.fq.gz flu_dIJ_cef_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEcef-4/19606△IJfluEcef-4_2.fq.gz flu_dIJ_cef_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEcipro-3/19606△IJfluEcipro-3_1.fq.gz flu_dIJ_cipro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEcipro-3/19606△IJfluEcipro-3_2.fq.gz flu_dIJ_cipro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEdori-1/19606△IJfluEdori-1_1.fq.gz flu_dIJ_dori_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEdori-1/19606△IJfluEdori-1_2.fq.gz flu_dIJ_dori_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEnitro-3/19606△IJfluEnitro-3_1.fq.gz flu_dIJ_nitro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEnitro-3/19606△IJfluEnitro-3_2.fq.gz flu_dIJ_nitro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEpip-4/19606△IJfluEpip-4_1.fq.gz flu_dIJ_pip_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEpip-4/19606△IJfluEpip-4_2.fq.gz flu_dIJ_pip_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEpolyB-4/19606△IJfluEpolyB-4_1.fq.gz flu_dIJ_polyB_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606△IJfluEpolyB-4/19606△IJfluEpolyB-4_2.fq.gz flu_dIJ_polyB_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEcef-1/19606wtfluEcef-1_1.fq.gz flu_wt_cef_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEcef-1/19606wtfluEcef-1_2.fq.gz flu_wt_cef_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEcipro-2/19606wtfluEcipro-2_1.fq.gz flu_wt_cipro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEcipro-2/19606wtfluEcipro-2_2.fq.gz flu_wt_cipro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEdori-1/19606wtfluEdori-1_1.fq.gz flu_wt_dori_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEdori-1/19606wtfluEdori-1_2.fq.gz flu_wt_dori_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEnitro-1/19606wtfluEnitro-1_1.fq.gz flu_wt_nitro_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEnitro-1/19606wtfluEnitro-1_2.fq.gz flu_wt_nitro_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEpip-4/19606wtfluEpip-4_1.fq.gz flu_wt_pip_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEpip-4/19606wtfluEpip-4_2.fq.gz flu_wt_pip_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEpolyB-4/19606wtfluEpolyB-4_1.fq.gz flu_wt_polyB_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEpolyB-4/19606wtfluEpolyB-4_2.fq.gz flu_wt_polyB_R2.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEtet-2/19606wtfluEtet-2_1.fq.gz flu_wt_tet_R1.fastq.gz
 ln -s ../../X101SC25116512-Z01-J003/01.RawData/19606wtfluEtet-2/19606wtfluEtet-2_2.fq.gz flu_wt_tet_R2.fastq.gz

 # ── Mitomycin C Dataset ──
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_cipro_1/MitoC_E_AB_cipro_1_1.fq.gz mito_dAB_cipro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_cipro_1/MitoC_E_AB_cipro_1_2.fq.gz mito_dAB_cipro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_dori_1/MitoC_E_AB_dori_1_1.fq.gz mito_dAB_dori_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_dori_1/MitoC_E_AB_dori_1_2.fq.gz mito_dAB_dori_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_nitro_2/MitoC_E_AB_nitro_2_1.fq.gz mito_dAB_nitro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_nitro_2/MitoC_E_AB_nitro_2_2.fq.gz mito_dAB_nitro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_tet_2/MitoC_E_AB_tet_2_1.fq.gz mito_dAB_tet_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_tet_2/MitoC_E_AB_tet_2_2.fq.gz mito_dAB_tet_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_trime_4/MitoC_E_AB_trime_4_1.fq.gz mito_dAB_trime_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_AB_trime_4/MitoC_E_AB_trime_4_2.fq.gz mito_dAB_trime_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_cipro_1/MitoC_E_IJ_cipro_1_1.fq.gz mito_dIJ_cipro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_cipro_1/MitoC_E_IJ_cipro_1_2.fq.gz mito_dIJ_cipro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_dori_4/MitoC_E_IJ_dori_4_1.fq.gz mito_dIJ_dori_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_dori_4/MitoC_E_IJ_dori_4_2.fq.gz mito_dIJ_dori_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_nitro_2/MitoC_E_IJ_nitro_2_1.fq.gz mito_dIJ_nitro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_nitro_2/MitoC_E_IJ_nitro_2_2.fq.gz mito_dIJ_nitro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_polyB_3/MitoC_E_IJ_polyB_3_1.fq.gz mito_dIJ_polyB_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_polyB_3/MitoC_E_IJ_polyB_3_2.fq.gz mito_dIJ_polyB_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_tet_3/MitoC_E_IJ_tet_3_1.fq.gz mito_dIJ_tet_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_tet_3/MitoC_E_IJ_tet_3_2.fq.gz mito_dIJ_tet_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_trime_1/MitoC_E_IJ_trime_1_1.fq.gz mito_dIJ_trime_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_IJ_trime_1/MitoC_E_IJ_trime_1_2.fq.gz mito_dIJ_trime_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_cipro_1/MitoC_E_wt_cipro_1_1.fq.gz mito_wt_cipro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_cipro_1/MitoC_E_wt_cipro_1_2.fq.gz mito_wt_cipro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_nitro_1/MitoC_E_wt_nitro_1_1.fq.gz mito_wt_nitro_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_nitro_1/MitoC_E_wt_nitro_1_2.fq.gz mito_wt_nitro_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_polyB_1/MitoC_E_wt_polyB_1_1.fq.gz mito_wt_polyB_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_polyB_1/MitoC_E_wt_polyB_1_2.fq.gz mito_wt_polyB_R2.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_trime_2/MitoC_E_wt_trime_2_1.fq.gz mito_wt_trime_R1.fastq.gz
 ln -s ../../X101SC26025981-Z02-J002/01.RawData/MitoC_E_wt_trime_2/MitoC_E_wt_trime_2_2.fq.gz mito_wt_trime_R2.fastq.gz

Call variant calling using snippy

 ln -s ~/Tools/bacto/db/ .;
 ln -s ~/Tools/bacto/envs/ .;
 ln -s ~/Tools/bacto/local/ .;
 cp ~/Tools/bacto/Snakefile .;
 cp ~/Tools/bacto/bacto-0.1.json .;
 cp ~/Tools/bacto/cluster.json .;

 #download CP059040.gb from GenBank
 #mv ~/Downloads/sequence\(2\).gb db/CP059040.gb

 mamba activate /home/jhuang/miniconda3/envs/bengal3_ac3
 (bengal3_ac3) jhuang@WS-2290C:~/DATA/Data_Tam_DNAseq_2023_A6WT_A10CraA_A12AYE_A1917978$ which snakemake
 /home/jhuang/miniconda3/envs/bengal3_ac3/bin/snakemake
 (bengal3_ac3) jhuang@WS-2290C:~/DATA/Data_Tam_DNAseq_2023_A6WT_A10CraA_A12AYE_A1917978$ snakemake -v
 4.0.0 --> CORRECT!

 #NOTE_1: modify bacto-0.1.json keeping only steps assembly, typing_mlst, possibly pangenome and variants_calling true; setting cpu=20 in all used steps.
     #setting the following in bacto-0.1.json
     "fastqc": false,
     "taxonomic_classifier": false,
     "assembly": true,
     "typing_ariba": false,
     "typing_mlst": true,
     "pangenome": true,
     "variants_calling": true,
     "phylogeny_fasttree": false,
     "phylogeny_raxml": false,
     "recombination": false, (due to gubbins-error set false)

     "prokka": {
       "genus": "Acinetobacter",
       "kingdom": "Bacteria",
       "species": "baumannii",
       "cpu": 10,
       "evalue": "1e-06",
       "other": ""
     },

     "mykrobe": {
       "species": "abaum"
     },

     "reference": "db/CP059040.gb"

 #NOTE_2: needs disk Titisee since the pipeline needs /media/jhuang/Titisee/GAMOLA2/TIGRfam_db/TIGRFAMs_15.0_HMM.LIB
 snakemake --printshellcmds

Summarize all SNPs and Indels from the snippy result directory.

 cp ~/Scripts/summarize_snippy_res_ordered.py .
 # IMPORTANT_ADAPT the array in script should be adapted; deleting the isolates "flu_wt_cipro" and "flu_dAB_cipro"
 isolates = ["flu_wt_cef", "flu_wt_dori", "flu_wt_nitro", "flu_wt_pip", "flu_wt_polyB", "flu_wt_tet",    "flu_dAB_cef", "flu_dAB_dori", "flu_dAB_nitro", "flu_dAB_pip", "flu_dAB_polyB", "flu_dAB_tet",    "flu_dIJ_cef", "flu_dIJ_cipro", "flu_dIJ_dori", "flu_dIJ_nitro", "flu_dIJ_pip", "flu_dIJ_polyB",         "mito_dIJ_trime",    "mito_wt_cipro", "mito_wt_nitro", "mito_wt_polyB", "mito_wt_trime",    "mito_dAB_cipro", "mito_dAB_dori", "mito_dAB_nitro", "mito_dAB_tet", "mito_dAB_trime",    "mito_dIJ_cipro", "mito_dIJ_dori", "mito_dIJ_nitro", "mito_dIJ_polyB", "mito_dIJ_tet"]

 mamba activate plot-numpy1
 python3 ./summarize_snippy_res_ordered.py snippy
 #--> Summary CSV file created successfully at: snippy/summary_snps_indels.csv
 cd snippy
 #REMOVE_the_line? I don't find the sence of the line:    grep -v "None,,,,,,None,None" summary_snps_indels.csv > summary_snps_indels_.csv

Using spandx calling variants (almost the same results to the one from viral-ngs!)

 mamba deactivate
 mamba activate /home/jhuang/miniconda3/envs/spandx

 # PREPARE the inputs for the options ref and database
 mkdir ~/miniconda3/envs/spandx/share/snpeff-5.1-2/data/PP810610
 cp PP810610.gb  ~/miniconda3/envs/spandx/share/snpeff-5.1-2/data/PP810610/genes.gbk
 vim ~/miniconda3/envs/spandx/share/snpeff-5.1-2/snpEff.config
 /home/jhuang/miniconda3/envs/spandx/bin/snpEff build PP810610    #-d
 ~/Scripts/genbank2fasta.py PP810610.gb
 mv PP810610.gb_converted.fna PP810610.fasta    #rename "NC_001348.1 xxxxx" to "NC_001348" in the fasta-file

 ln -s /home/jhuang/Tools/spandx/ spandx
 # Deleting the contaminated samples flu_wt_cipro*.fastq and flu_dAB_cipro*.fastq
 (spandx) nextflow run spandx/main.nf --fastq "trimmed/*_P_{1,2}.fastq" --ref CP059040.fasta --annotation --database CP059040 -resume

 # RERUN SNP_matrix.sh due to the error ERROR_CHROMOSOME_NOT_FOUND in the variants annotation, resulting in all impacts are MODIFIER --> IT WORKS!
 cd Outputs/Master_vcf
 conda activate /home/jhuang/miniconda3/envs/spandx
 (spandx) cp -r ../../snippy/Y1/reference . # Eigentlich irgendein directory, all directories contains the same reference.
 (spandx) cp ../../spandx/bin/SNP_matrix.sh ./
 #Note that ${variant_genome_path}=CP059040 in the following command, but it was not used after the following command modification.
 #Adapt "snpEff eff -no-downstream -no-intergenic -ud 100 -formatEff -v ${variant_genome_path} out.vcf > out.annotated.vcf" to
 "/home/jhuang/miniconda3/envs/bengal3_ac3/bin/snpEff eff -no-downstream -no-intergenic -ud 100 -formatEff -c reference/snpeff.config -dataDir . ref out.vcf > out.annotated.vcf" in SNP_matrix.sh
 (spandx) bash SNP_matrix.sh CP059040 .

Cross-Caller SNP/Indel Concordance & Invariant Variant Analyzer; Multi-Isolate Variant Intersection, Annotation Harmonization & Caller Discrepancy Report; Comparative Genomic Variant Profiling: Concordance, Invariance & Allele Mismatch Analysis; VarMatch: Cross-Tool Variant Concordance Pipeline

Calling inter-host variants by merging the results from snippy+spandx

 mamba activate plot-numpy1
 cd bacto
 cp Outputs/Master_vcf/All_SNPs_indels_annotated.txt .
 cp snippy/summary_snps_indels.csv .

 cp ~/Scripts/process_variants_snippy_alleles_spandx_annotations.py .

 #Configuring
 ISOLATES = [
         "flu_wt_cef", "flu_wt_cipro", "flu_wt_dori", "flu_wt_nitro", "flu_wt_pip", "flu_wt_polyB", "flu_wt_tet",
         "flu_dAB_cef", "flu_dAB_cipro", "flu_dAB_dori", "flu_dAB_nitro", "flu_dAB_pip", "flu_dAB_polyB", "flu_dAB_tet",
         "flu_dIJ_cef", "flu_dIJ_cipro", "flu_dIJ_dori", "flu_dIJ_nitro", "flu_dIJ_pip", "flu_dIJ_polyB",
         "mito_dIJ_trime",
         "mito_wt_cipro", "mito_wt_nitro", "mito_wt_polyB", "mito_wt_trime",
         "mito_dAB_cipro", "mito_dAB_dori", "mito_dAB_nitro", "mito_dAB_tet", "mito_dAB_trime",
         "mito_dIJ_cipro", "mito_dIJ_dori", "mito_dIJ_nitro", "mito_dIJ_polyB", "mito_dIJ_tet"
         ]

 (plot-numpy1) python process_variants_snippy_alleles_spandx_annotations.py

 # mv common_variants_all_snippy_annotated.xlsx common_variants_snippy+spandx_annotated_19606wt_dAB_dIJ_mito_flu.xlsx
 # mv common_variants_invariant_snippy_annotated.xlsx common_invariants_snippy+spandx_annotated_19606wt_dAB_dIJ_mito_flu.xlsx

(TODO_TOMORROW) Manully checking each of the 6 records by comparing them to the results from SPANDx; three are confirmed!

 #CHROM,POS,REF,ALT,TYPE,Y1,Y2,Y3,Y4,W1,W2,W3,W4,Effect,Impact,Functional_Class,Codon_change,Protein_and_nucleotide_change,Amino_Acid_Length,Gene_name,Biotype

 # -- Results from snippy --
 #move: CP059040,1527276,TTGAACC,T,del,TTGAACC,TTGAACC,TTGAACC,T,TTGAACC,TTGAACC,T,T,conservative_inframe_deletion,MODERATE,,gaacct/,p.Glu443_Pro444del/c.1327_1332delGAACCT,704,H0N29_07175,protein_coding
 #confirmed: CP059040,1843289,G,T,snp,G,T,G,G,G,G,G,G,missense_variant,MODERATE,MISSENSE,gCg/gAg,p.Ala37Glu/c.110C>A,357,H0N29_08665,protein_coding
 #confirmed: CP059040,2019186,G,A,snp,A,G,G,G,G,G,G,G,upstream_gene_variant,MODIFIER,,59,c.-59C>T,144,H0N29_09480,protein_coding
 #delete_this? CP059040,3124917,T,"TAC,TACTTCATTACATACCAACCGCCAAGGGTGC",snp,C,T,C,C,T,T,T,C,upstream_gene_variant,MODIFIER,,25,c.-25_-24insAC,0,H0N29_00075,protein_coding
 #move: CP059040,3310021,C,CT,ins,CT,CT,CT,CT,C,CT,CT,CT,intragenic_variant,MODIFIER,,,n.3310021_3310022insT,,H0N29_00075,
 #confirmed: CP059040,3853714,G,A,snp,G,G,G,G,G,A,G,A,stop_gained,HIGH,NONSENSE,Cag/Tag,p.Gln91*/c.271C>T,338,H0N29_18290,protein_coding
 #--> Only three SNPs are confirmed --> means there is almost no variation in the genomic level!

 # -- Results from the SPANDx --
 #CP059040   1527276 TTGAACC T   INDEL   TTGAACC/T   T   T   T   T   T   T   T   conservative_inframe_deletion   MODERATE        gaacct/ p.Glu443_Pro444del/c.1327_1332delGAACCT 704 H0N29_07175 protein_coding

 #CP059040   1843289 G   T   SNP G   T   G   G   G   G   G   G   missense_variant    MODERATE    MISSENSE    gCg/gAg p.Ala37Glu/c.110C>A 357 H0N29_08665 protein_coding
 #CP059040   2019186 G   A   SNP A   G   G   G   G   G   G   G   upstream_gene_variant   MODIFIER        59  c.-59C>T    144 H0N29_09480 protein_coding

 #Cmp to CP059040    3124917 T   TAC,TACTTCATTACATACCAACCGCCAAGGGTGC INDEL   .   TACTTCATTACATACCAACCGCCAAGGGTGC TACTTCATTACATACCAACCGCCAAGGGTGC TAC .   .   .   .   upstream_gene_variant   MODIFIER        25  c.-25_-24insAC  0   H0N29_00075 protein_coding
 #Cmp to CP059040    3124920 C   CATTACATACCAACCGCCAAGGGTGCTTCATG    INDEL   .   .   .   CATTACATACCAACCGCCAAGGGTGCTTCATG    .   .   C   .   upstream_gene_variant   MODIFIER        22  c.-22_-21insATTACATACCAACCGCCAAGGGTGCTTCATG 0   H0N29_00075 protein_coding

 #TODO: Move to invariant-file: CP059040 3310021 C   CT  INDEL   CT  CT  CT  CT  CT  CT  CT  CT  intragenic_variant  MODIFIER            n.3310021_3310022insT       H0N29_00075
 #CP059040   3853714 G   A   SNP G   G   G   G   G   A   G   A   stop_gained HIGH    NONSENSE    Cag/Tag p.Gln91*/c.271C>T   338 H0N29_18290 protein_coding

 #-->For the Indel-report, more complicated, needs the following command to find the initial change and related codon-change.
 ## Check gene strand in your GFF/GenBank
 #grep "H0N29_07175" reference.gff
 # Extract 20 bp around the variant from reference
 samtools faidx CP059040.fasta CP059040:1527260-1527290

Run nextflow bacass

 # Download the kmerfinder database: https://www.genomicepidemiology.org/services/ --> https://cge.food.dtu.dk/services/KmerFinder/ --> https://cge.food.dtu.dk/services/KmerFinder/etc/kmerfinder_db.tar.gz
 # Download 20190108_kmerfinder_stable_dirs.tar.gz from https://zenodo.org/records/13447056
 #--kmerfinderdb /mnt/nvme1n1p1/REFs/kmerfinder_db.tar.gz
 #--kmerfinderdb /mnt/nvme1n1p1/REFs/20190108_kmerfinder_stable_dirs.tar.gz
 nextflow run nf-core/bacass -r 2.5.0 -profile docker \
 --input samplesheet.tsv \
 --outdir bacass_out \
 --assembly_type short \
 --kraken2db /mnt/nvme1n1p1/REFs/k2_standard_08_GB_20251015.tar.gz \
 --kmerfinderdb /mnt/nvme1n1p1/REFs/kmerfinder/bacteria/ \
 -resume
 #Possibly the chraracter '△' is a problem.
 #Solution: 19606△ABfluEcef-1 → 19606delABfluEcef-1

 #SAVE bacass_out/Kmerfinder/kmerfinder_summary.csv to bacass_out/Kmerfinder/An6?/An6?_kmerfinder_results.xlsx

 samplesheet.tsv
 sample,fastq_1,fastq_2
 flu_dAB_cef,../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcef-1/19606△ABfluEcef-1_1.fq.gz,../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcef-1/19606△ABfluEcef-1_2.fq.gz
 flu_dAB_cipro,../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcipro-2/19606△ABfluEcipro-2_1.fq.gz,../X101SC25116512-Z01-J003/01.RawData/19606△ABfluEcipro-2/19606△ABfluEcipro-2_2.fq.gz

 #busco example results:
 Input_file      Dataset Complete        Single  Duplicated      Fragmented      Missing n_markers       Scaffold N50    Contigs N50     Percent gaps    Number of scaffolds
 wt_cef.scaffolds.fa     bacteria_odb10  98.4    98.4    0.0     1.6     0.0     124     285852  285852  0.000%  45
 wt_cipro.scaffolds.fa   bacteria_odb10  90.3    89.5    0.8     8.1     1.6     124     7434    7434    0.000%  1699

Run bactmap

 nextflow run nf-core/bactmap -r 1.0.0 -profile docker \
 --input samplesheet.csv \
 --reference CP059040.fasta \
 --outdir bactmap_out \
 -resume

 nextflow run nf-core/bactmap -r 1.0.0 -profile docker \
 --input samplesheet.csv \
 --reference CU459141.fasta \
 --outdir bactmap_out \
 -resume

 sample,fastq_1,fastq_2
 G18582004,fastqs/G18582004_1.fastq.gz,fastqs/G18582004_2.fastq.gz
 G18756254,fastqs/G18756254_1.fastq.gz,fastqs/G18756254_2.fastq.gz
 G18582006,fastqs/G18582006_1.fastq.gz,fastqs/G18582006_2.fastq.gz

 mkdir bactmap_workspace
 #Prepare reference.fasta (example for CP059040.fasta) in bactmap_workspace and samplesheet.csv in bactmap_workspace
 nextflow run nf-core/bactmap -r 1.0.0 -profile docker \
 --input samplesheet.csv \
 --reference CP059040.fasta \
 --outdir bactmap_out \
 -resume

Microbial bioinformatics

Microbial bioinformatics uses computational tools to analyze genomes, track evolution, and study functions in microorganisms, including bacteria and viruses.

Interhost variant calling (Data_Tam_DNAseq_2026_19606wt_dAB_dIJ_mito_flu_on_ATCC19606)

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