Calling inter-host variants by merging the results from snippy+spandx (Manually!) Calling intra-host variants using viral-ngs (http://xgenes.com/article/article-content/347/variant-calling-for-herpes-simplex-virus-1-from-patient-sample-using-capture-probe-sequencing/) #TODO: How? Merge intra- and inter-host variants, comparing the variants to the alignments of the assemblies to confirm its correctness.
#TODO: If the results from 2024 contains only intra-host variants, explain this time I also give the results of the inter-host variants!
Variant calling (inter-host + intra-host) for Data_Pietschmann_229ECoronavirus_Mutations_2024+2025+2026 (via docker own_viral_ngs) v2
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Input data:
# ---- Datasets 2024 (in total 4) ---- ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/hCoV229E_Rluc_R1.fastq.gz hCoV229E_Rluc_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/hCoV229E_Rluc_R2.fastq.gz hCoV229E_Rluc_R2.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_DMSO_R1.fastq.gz DMSO_p10_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_DMSO_R2.fastq.gz DMSO_p10_R2.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_K22_R1.fastq.gz K22_p10_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_K22_R2.fastq.gz K22_p10_R2.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_K7523_R1.fastq.gz X7523_p10_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2024/p10_K7523_R2.fastq.gz X7523_p10_R2.fastq.gz # ---- Datasets 2025 (in total 3) ---- ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20606/p16_DMSO_S29_R1_001.fastq.gz DMSO_p16_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20606/p16_DMSO_S29_R2_001.fastq.gz DMSO_p16_R2.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20607/p16_K22_S30_R1_001.fastq.gz K22_p16_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20607/p16_K22_S30_R2_001.fastq.gz K22_p16_R2.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20608/p16_X7523_S31_R1_001.fastq.gz X7523_p16_R1.fastq.gz ln -s ../../Data_Pietschmann_229ECoronavirus_Mutations_2025/raw_data_2025/250506_VH00358_136_AAG3YJ5M5/p20608/p16_X7523_S31_R2_001.fastq.gz X7523_p16_R2.fastq.gz # ---- Datasets 2026 (in total 3) ---- ln -s ../raw_data_2026/20260212_AV243904_0054_B/02_DMSO_p26/02_DMSO_p26_R1.fastq.gz DMSO_p26_R1.fastq.gz ln -s ../raw_data_2026/20260212_AV243904_0054_B/02_DMSO_p26/02_DMSO_p26_R2.fastq.gz DMSO_p26_R2.fastq.gz ln -s ../raw_data_2026/20260212_AV243904_0054_B/01_K22_p26/01_K22_p26_R1.fastq.gz K22_p26_R1.fastq.gz ln -s ../raw_data_2026/20260212_AV243904_0054_B/01_K22_p26/01_K22_p26_R2.fastq.gz K22_p26_R2.fastq.gz ln -s ../raw_data_2026/20260212_AV243904_0054_B/03_X723_p26/03_X723_p26_R1.fastq.gz X7523_p26_R1.fastq.gz ln -s ../raw_data_2026/20260212_AV243904_0054_B/03_X723_p26/03_X723_p26_R2.fastq.gz X7523_p26_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 CU459141.gb from GenBank mv ~/Downloads/sequence\(2\).gb db/PP810610.gb #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": true, "phylogeny_raxml": true, "recombination": false, (due to gubbins-error set false) "genus": "Alphacoronavirus", "kingdom": "Viruses", "species": "Human coronavirus 229E", "mykrobe": { "species": "corona" }, "reference": "db/PP810610.gb" mamba activate /home/jhuang/miniconda3/envs/bengal3_ac3 (bengal3_ac3) /home/jhuang/miniconda3/envs/snakemake_4_3_1/bin/snakemake --printshellcmds -
Summarize all SNPs and Indels from the snippy result directory.
cp ~/Scripts/summarize_snippy_res_ordered.py . # IMPORTANT_ADAPT the array isolates = ["hCoV229E_Rluc", "DMSO_p10", "K22_p10", "X7523_p10", "DMSO_p16", "K22_p16", "X7523_p16", "DMSO_p26", "K22_p26", "X7523_p26"] 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 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 (spandx) nextflow run spandx/main.nf --fastq "trimmed/*_P_{1,2}.fastq" --ref PP810610.fasta --annotation --database PP810610 -resume # Rerun SNP_matrix.sh due to the error ERROR_CHROMOSOME_NOT_FOUND in the variants annotation cd Outputs/Master_vcf (spandx) cp -r ../../snippy/hCoV229E_Rluc/reference . (spandx) cp ../../spandx/bin/SNP_matrix.sh ./ #Note that ${variant_genome_path}=NC_001348 in the following command, but it was not used after command replacement. #Adapt "snpEff eff -no-downstream -no-intergenic -ud 100 -formatEff -v ${variant_genome_path} out.vcf > out.annotated.vcf" to "/home/jhuang/miniconda3/envs/bengal3_ac3/bin/snpEff eff -no-downstream -no-intergenic -ud 100 -formatEff -c reference/snpeff.config -dataDir . ref out.vcf > out.annotated.vcf" in SNP_matrix.sh (spandx) bash SNP_matrix.sh PP810610 . -
Calling inter-host variants by merging the results from snippy+spandx (Manually!)
# Inter-host variants（宿主间变异）:一种病毒在两个人之间有不同的基因变异，这些变异可能与宿主的免疫反应、疾病表现或病毒传播的方式相关。 cp All_SNPs_indels_annotated.txt All_SNPs_indels_annotated_backup.txt vim All_SNPs_indels_annotated.txt #in the file ids: grep "$(echo -e '\t')353$(echo -e '\t')" All_SNPs_indels_annotated.txt >> All_SNPs_indels_annotated_.txt #Replace \n with " All_SNPs_indels_annotated.txt >> All_SNPs_indels_annotated_.txt\ngrep " #Replace grep " --> grep "$(echo -e '\t') #Replace " All_ --> $(echo -e '\t')" All_ # Potential intra-host variants: 10871, 19289, 23435. CHROM POS REF ALT TYPE hCoV229E_Rluc_trimmed p10_DMSO_trimmed p10_K22_trimmed p10_K7523_trimmed p16_DMSO_trimmed p16_K22_trimmed p16_X7523_trimmed Effect Impact Functional_Class Codon_change Protein_and_nucleotide_change Amino_Acid_Length Gene_name Biotype PP810610 1464 T C SNP C C C C C C C missense_variant MODERATE MISSENSE gTt/gCt p.Val416Ala/c.1247T>C 6757 CDS_1 protein_coding PP810610 1699 C T SNP T T T T T T T synonymous_variant LOW SILENT gtC/gtT p.Val494Val/c.1482C>T 6757 CDS_1 protein_coding PP810610 6691 C T SNP T T T T T T T synonymous_variant LOW SILENT tgC/tgT p.Cys2158Cys/c.6474C>T 6757 CDS_1 protein_coding PP810610 6919 C G SNP G G G G G G G synonymous_variant LOW SILENT ggC/ggG p.Gly2234Gly/c.6702C>G 6757 CDS_1 protein_coding PP810610 7294 T A SNP A A A A A A A missense_variant MODERATE MISSENSE agT/agA p.Ser2359Arg/c.7077T>A 6757 CDS_1 protein_coding * PP810610 10871 C T SNP C C/T T C/T C/T T C/T missense_variant MODERATE MISSENSE Ctt/Ttt p.Leu3552Phe/c.10654C>T 6757 CDS_1 protein_coding PP810610 14472 T C SNP C C C C C C C missense_variant MODERATE MISSENSE aTg/aCg p.Met4752Thr/c.14255T>C 6757 CDS_1 protein_coding PP810610 15458 T C SNP C C C C C C C synonymous_variant LOW SILENT Ttg/Ctg p.Leu5081Leu/c.15241T>C 6757 CDS_1 protein_coding PP810610 16035 C A SNP A A A A A A A stop_gained HIGH NONSENSE tCa/tAa p.Ser5273*/c.15818C>A 6757 CDS_1 protein_coding PP810610 17430 T C SNP C C C C C C C missense_variant MODERATE MISSENSE tTa/tCa p.Leu5738Ser/c.17213T>C 6757 CDS_1 protein_coding * PP810610 19289 G T SNP G G T G G G/T G missense_variant MODERATE MISSENSE Gtt/Ttt p.Val6358Phe/c.19072G>T 6757 CDS_1 protein_coding PP810610 21183 T G SNP G G G G G G G missense_variant MODERATE MISSENSE tTt/tGt p.Phe230Cys/c.689T>G 1173 CDS_2 protein_coding PP810610 22636 T G SNP G G G G G G G missense_variant MODERATE MISSENSE aaT/aaG p.Asn714Lys/c.2142T>G 1173 CDS_2 protein_coding PP810610 23022 T C SNP C C C C C C C missense_variant MODERATE MISSENSE tTa/tCa p.Leu843Ser/c.2528T>C 1173 CDS_2 protein_coding * PP810610 23435 C T SNP C C T C/T C C/T C/T missense_variant MODERATE MISSENSE Ctt/Ttt p.Leu981Phe/c.2941C>T 1173 CDS_2 protein_coding PP810610 24512 C T SNP T T T T T T T missense_variant MODERATE MISSENSE Ctc/Ttc p.Leu36Phe/c.106C>T 88 CDS_4 protein_coding PP810610 24781 C T SNP T T T T T T T missense_variant MODERATE MISSENSE aCt/aTt p.Thr36Ile/c.107C>T 77 CDS_5 protein_coding PP810610 25163 C T SNP T T T T T T T missense_variant MODERATE MISSENSE Ctt/Ttt p.Leu82Phe/c.244C>T 225 CDS_6 protein_coding PP810610 25264 C T SNP T T T T T T T synonymous_variant LOW SILENT gtC/gtT p.Val115Val/c.345C>T 225 CDS_6 protein_coding PP810610 26838 G T SNP T T T T T T T -
Calling intra-host variants using viral-ngs
# Intra-host variants（宿主内变异）：同一个人感染了某种病毒，但在其体内的不同细胞或器官中可能存在多个不同的病毒变异株。 #How to run and debug the viral-ngs docker? # ---- DEBUG_2026_1: using docker instead ---- mkdir viralngs; cd viralngs ln -s ~/Tools/viral-ngs_docker/Snakefile Snakefile ln -s ~/Tools/viral-ngs_docker/bin bin cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/refsel.acids refsel.acids cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/lastal.acids lastal.acids cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/config.yaml config.yaml cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/samples-runs.txt samples-runs.txt cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/samples-depletion.txt samples-depletion.txt cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/samples-metagenomics.txt samples-metagenomics.txt cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/samples-assembly.txt samples-assembly.txt cp ~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2024/samples-assembly-failures.txt samples-assembly-failures.txt # Adapt the sample-*.txt mkdir viralngs/data mkdir viralngs/data/00_raw mkdir bams ref_fa="PP810610.fasta"; #for sample in hCoV229E_Rluc p10_DMSO p10_K22; do #for sample in p10_K7523 p16_DMSO p16_K22 p16_X7523; do for sample in hCoV229E_Rluc DMSO_p10 K22_p10 X7523_p10 DMSO_p16 K22_p16 X7523_p16 DMSO_p26 K22_p26 X7523_p26; do bwa index ${ref_fa}; \ bwa mem -M -t 16 ${ref_fa} trimmed/${sample}_trimmed_P_1.fastq trimmed/${sample}_trimmed_P_2.fastq | samtools view -bS - > bams/${sample}_genome_alignment.bam; \ done conda activate viral-ngs4 #for sample in hCoV229E_Rluc p10_DMSO p10_K22; do #for sample in p10_K7523 p16_DMSO p16_K22 p16_X7523; do for sample in hCoV229E_Rluc DMSO_p10 K22_p10 X7523_p10 DMSO_p16 K22_p16 X7523_p16 DMSO_p26 K22_p26 X7523_p26; do picard AddOrReplaceReadGroups I=bams/${sample}_genome_alignment.bam O=~/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2026/viralngs/data/00_raw/${sample}.bam SORT_ORDER=coordinate CREATE_INDEX=true RGPL=illumina RGID=$sample RGSM=$sample RGLB=standard RGPU=$sample VALIDATION_STRINGENCY=LENIENT; \ done conda deactivate # -- ! Firstly set the samples-assembly.txt empty, so that only focus on running depletion! docker run -it -v /mnt/md1/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2026/viralngs:/work -v /home/jhuang/Tools/viral-ngs_docker:/home/jhuang/Tools/viral-ngs_docker -v /home/jhuang/REFs:/home/jhuang/REFs -v /home/jhuang/Tools/GenomeAnalysisTK-3.6:/home/jhuang/Tools/GenomeAnalysisTK-3.6 -v /home/jhuang/Tools/novocraft_v3:/home/jhuang/Tools/novocraft_v3 -v /usr/local/bin/gatk:/usr/local/bin/gatk own_viral_ngs bash cd /work snakemake --directory /work --printshellcmds --cores 80 # -- ! Secondly manully run assembly steps # --> By itereative add the unfinished assembly in the list, each time replace one, and run "cd /work; snakemake --directory /work --printshellcmds --cores 80" after exiting and re-entering the docker-env, since some tools were during the running automatically deleted.
Here is the combined, consolidated fix sequence for your Docker container:
🔧 Complete Fix Commands (Run Inside Docker Container)
#!/bin/bash
# ============================================================
# FIX SCRIPT FOR viral-ngs Docker Environment
# Run these commands INSIDE your running Docker container
# ============================================================
echo "=== Step 1: Activate base environment for config changes ==="
conda activate base
echo "=== Step 2: Disable conda safety checks (fixes 'unsafe path' errors) ==="
conda config --set safety_checks disabled
conda config --set allow_softlinks true
echo "=== Step 3: Verify config was applied ==="
conda config --show safety_checks
# Expected output: safety_checks: disabled
echo "=== Step 4: (Optional) Update conda for compatibility ==="
conda update -n base -c defaults conda -y
echo "=== Step 5: Activate viral-ngs environment ==="
conda activate viral-ngs-env
echo "Current env: $CONDA_DEFAULT_ENV" && which python
#Current env: viral-ngs-env
#/opt/miniconda/bin/python --> ⚠️ Problem Identified!
#conda deactivate; conda deactivate;
## 1. Install mamba in your base environment (one-time setup)
#conda install -n base -c conda-forge mamba -y
## 2. Activate your existing environment
#conda activate viral-ngs-env
## 3. Use mamba to install missing packages (instead of conda)
#mamba install -c bioconda biopython mafft -y
## 4. Verify existing tools are still there
#which python
#conda list # Shows all packages, both old and new
conda install python=3.6.7 -y
which python
echo "Current env: $CONDA_DEFAULT_ENV" && which python
#Current env: viral-ngs-env
#/opt/miniconda/envs/viral-ngs-env/bin/python (Python 3.6.7)
echo "=== Step 6: Install missing Python packages ==="
conda install -y -c conda-forge biopython
echo "=== Step 7: Install missing binary tools (with specific versions if needed) ==="
conda install -y -c bioconda perl=5.32.1 prinseq-lite samtools
echo "=== Step 8: Verify all installations ==="
echo "--- Checking samtools ---"
which samtools && samtools --version
#/opt/miniconda/envs/viral-ngs-env/bin/samtools samtools 1.9 using htslib 1.9
echo "--- Checking perl ---"
which perl && perl --version
echo "--- Checking prinseq-lite ---"
which prinseq-lite.pl && prinseq-lite.pl -version
echo "--- Checking Biopython ---"
python -c "import Bio; print('Biopython OK:', Bio.__version__)"
echo "=== Step 9: Refresh environment PATH ==="
hash -r
echo "=== ✅ All fixes applied! You can now re-run your pipeline ==="
echo "Tip: Run 'snakemake --unlock' first if pipeline is locked, then:"
echo " snakemake -j
<threads> --rerun-incomplete"In Dockerfile
#ENV CONDA_ALLOW_UNSAFE_PATHS=1 #RUN conda update -n base -c defaults conda -y
🐳 To Make Fixes Permanent: Commit the Container
After running the fixes above, save your working container:
# 1. Exit the container (but don't delete it)
exit
docker ps -a
docker commit c51d44624f1b viral-ngs-fixed:2026-03-19
# 3. Next time, run the fixed image
#docker run -it -v /mnt/md1/... [your other volumes] viral-ngs-fixed:2026-03-19 bash
docker run -it -v /mnt/md1/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2026/viralngs:/work -v /home/jhuang/Tools/viral-ngs_docker:/home/jhuang/Tools/viral-ngs_docker -v /home/jhuang/REFs:/home/jhuang/REFs -v /home/jhuang/Tools/GenomeAnalysisTK-3.6:/home/jhuang/Tools/GenomeAnalysisTK-3.6 -v /home/jhuang/Tools/novocraft_v3:/home/jhuang/Tools/novocraft_v3 -v /usr/local/bin/gatk:/usr/local/bin/gatk viral-ngs-fixed:2026-03-19 bash
conda activate viral-ngs-env
which samtools && samtools --version
#/opt/miniconda/envs/viral-ngs-env/bin/samtools samtools 1.9 using htslib 1.9
which perl && perl --version
#/opt/miniconda/envs/viral-ngs-env/bin/perl (v5.26.2)
which prinseq-lite.pl && prinseq-lite.pl -version
#/opt/miniconda/envs/viral-ngs-env/bin/prinseq-lite.pl (PRINSEQ-lite 0.20.4)
python -c "import Bio; print('Biopython OK:', Bio.__version__)"
#Biopython OK: 1.72
conda install -c bioconda trimmomatic -y
which trimmomatic
#/opt/miniconda/envs/viral-ngs-env/bin/trimmomatic (trimmomatic-0.39)
exit
docker ps -a
docker commit e70395e5625c viral-ngs-fixed:l
docker run -it -v /mnt/md1/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2026/viralngs:/work -v /home/jhuang/Tools/viral-ngs_docker:/home/jhuang/Tools/viral-ngs_docker -v /home/jhuang/REFs:/home/jhuang/REFs -v /home/jhuang/Tools/GenomeAnalysisTK-3.6:/home/jhuang/Tools/GenomeAnalysisTK-3.6 -v /home/jhuang/Tools/novocraft_v3:/home/jhuang/Tools/novocraft_v3 -v /usr/local/bin/gatk:/usr/local/bin/gatk viral-ngs-fixed:l bash
cd /work
#!!!! IMPORTANT !!!!
conda activate viral-ngs-env
snakemake --directory /work --printshellcmds --cores 80#DEBUG the specific commmand as follows, for example install Gap2Seq in the docker-env. bin/assembly.py gapfill_gap2seq tmp/02_assembly/hCoV229E_Rluc.assembly2-scaffolded.fasta data/01_per_sample/hCoV229E_Rluc.cleaned.bam tmp/02_assembly/hCoV229E_Rluc.assembly2-gapfilled.fasta –memLimitGb 12 –maskErrors –randomSeed 0 —-> go to the script tools/gap2seq.py, install the required tool (for example gap2seq) and adapt the correct version. conda install -y -c bioconda gap2seq root@f47daf7c44ee:/work# Gap2Seq -h #>Gap2Seq 3.1 vim /home/jhuang/Tools/viral-ngs_docker/bin/tools/gap2seq.py #Adapt the TOOL_NAME and TOOL_VERSION in the script
#Save the docker-env with newly installed Gap2Seq exit docker ps -a docker commit f47daf7c44ee viral-ngs-fixed:la
#调用新的 docker-env installed with Gap2Seq docker run -it -v /mnt/md1/DATA_D/Data_Pietschmann_229ECoronavirus_Mutations_2026/viralngs:/work -v /home/jhuang/Tools/viral-ngs_docker:/home/jhuang/Tools/viral-ngs_docker -v /home/jhuang/REFs:/home/jhuang/REFs -v /home/jhuang/Tools/GenomeAnalysisTK-3.6:/home/jhuang/Tools/GenomeAnalysisTK-3.6 -v /home/jhuang/Tools/novocraft_v3:/home/jhuang/Tools/novocraft_v3 -v /usr/local/bin/gatk:/usr/local/bin/gatk viral-ngs-fixed:la bash cd /work conda activate viral-ngs-env snakemake –directory /work –printshellcmds –cores 80
#MANUALLY running the following commands! bin/assembly.py gapfill_gap2seq in_scaffold=tmp/02_assembly/hCoV229E_Rluc.assembly2-scaffolded.fasta in_bam=data/01_per_sample/hCoV229E_Rluc.cleaned.bam out_scaffold=tmp/02_assembly/hCoV229E_Rluc.assembly2-gapfilled.fasta mem_limit_gb=12 time_soft_limit_minutes=60.0 mask_errors=True gap2seq_opts= random_seed=0 threads=None loglevel=INFO tmp_dir=/tmp tmp_dirKeep=False
#MANUALLY running the following commands! bin/assembly.py impute_from_reference tmp/02_assembly/hCoV229E_Rluc.assembly2-gapfilled.fasta tmp/02_assembly/hCoV229E_Rluc.assembly2-scaffold_ref.fasta tmp/02_assembly/hCoV229E_Rluc.assembly3-modify.fasta –newName hCoV229E_Rluc –replaceLength 55 –minLengthFraction 0.05 –minUnambig 0.05 –index
#!!!! TODO_NEXT_WEEK !!!!: run several times of docker so that all ${sample}.assembly2-scaffolded.fasta generated! Then run the following commands, after that run docker for all isolates. for sample in DMSO_p10 K22_p10 X7523_p10 DMSO_p16 K22_p16 X7523_p16 DMSO_p26 K22_p26 X7523_p26; do #MANUALLY running the following commands! bin/assembly.py gapfill_gap2seq in_scaffold=tmp/02_assembly/${sample}.assembly2-scaffolded.fasta in_bam=data/01_per_sample/${sample}.cleaned.bam out_scaffold=tmp/02_assembly/${sample}.assembly2-gapfilled.fasta mem_limit_gb=12 time_soft_limit_minutes=60.0 mask_errors=True gap2seq_opts= random_seed=0 threads=None loglevel=INFO tmp_dir=/tmp tmp_dirKeep=False #MANUALLY running the following commands! bin/assembly.py impute_from_reference tmp/02_assembly/${sample}.assembly2-gapfilled.fasta tmp/02_assembly/${sample}.assembly2-scaffold_ref.fasta tmp/02_assembly/${sample}.assembly3-modify.fasta –newName ${sample} –replaceLength 55 –minLengthFraction 0.05 –minUnambig 0.05 –index done
🔍 Troubleshooting Checklist
If issues persist after running the fix:
# Check conda config
conda config --show | grep -E "safety|softlink"
# List installed packages in viral-ngs-env
conda activate viral-ngs-env
conda list | grep -E "samtools|prinseq|perl|biopython"
# Test each tool manually
samtools --version
prinseq-lite.pl -version
perl -e 'use Bio::Seq; print "BioPerl OK\n"'
# Check PATH includes conda bin directories
echo $PATH | tr ':' '\n' | grep conda⚠️ Important Notes
| Issue | Solution |
|---|---|
safety_checks disabled not working |
Must run conda config in base env, not viral-ngs-env |
| Packages still fail to install | Try conda clean --all -y first, then reinstall |
samtools: command not found after install |
Run hash -r or restart shell to refresh PATH |
| Pipeline still fails after fixes | Run snakemake --unlock --rerun-incomplete to resume |
conda config --set safety_checks disable
conda activate viral-ngs-env
conda install -y -c conda-forge biopython
docker ps -a
# Look for the container you are working in, e.g., "viral-ngs-container"
#bb117a6ca70a
docker commit