WeichenZhou / LIBD75

Codes for LIBD75 Project

LIBD75

Base-calling

Assembly

• Flye:

flye --nano-raw path-of-lr-fastq -g 3g -o flye -t 30

• Shasta:

./shasta-Linux-0.11.1 --input /nfs/turbo/umms-smaht/working/202407_ONT_merging/ONT.bulk.fastq --config customized.conf --assemblyDirectory shasta --threads 30

• Hapdup:

minimap2 -ax map-ont -t 30 $HD_DIR/flye/assembly.fasta path-of-lr-fastq | samtools sort -@ 4 -m 4G > $HD_DIR/hapdup/lr_mapping.bam
samtools index -@ 4 $HD_DIR/hapdup/lr_mapping.bam

singularity exec --bind $HD_DIR hapdup_0.12.sif hapdup --assembly $HD_DIR/flye/assembly.fasta --bam $HD_DIR/hapdup/lr_mapping.bam --out-dir hapdup -t 24 --rtype ont

• Hapog:

Alignment

Genetic Variant Calling

• MEI

  • Illumina

    • Single Cell

      • xTea version 0.1.9

      ./xTea/bin/xtea -i sample_id.txt -b illumina_bam_list.txt -x null -p $Work_dire -o $Work_dire/submit_jobs.sh -l $Rep_lib -r GCA_000001405.15_GRCh38_no_alt_analysis_set.fa -g gencode.v33.annotation.gff3 --xtea ./xTea/bin/xtea/ -f 5907 -y 7 --user --nclip 1 --cr 0 --nd 0
      
      # sample_id.txt: samples id list
      # illumina_bam_list.txt: Illumina bam file list
      
      

      • MELT version 2.2.2

      java -jar ./MELTv2.2.2/MELT.jar Single -h GCA_000001405.15_GRCh38_no_alt_analysis_set.fa -bamfile $bam_file -n ./MELTv2.2.2/add_bed_files/Hg38/Hg38.genes.bed -t ./MELTv2.2.2/me_refs/Hg38/mei_list.txt -w $result_dire
      

    • Bulk

      • xTea version 0.1.9

      ./xTea/bin/xtea -i sample_id.txt -b illumina_bam_list.txt -x null -p $Work_dire -o $Work_dire/submit_jobs.sh -l $Rep_lib -r GCA_000001405.15_GRCh38_no_alt_analysis_set.fa -g gencode.v33.annotation.gff3 --xtea ./xTea/bin/xtea/ -f 5907 -y 7
      # sample_id.txt: samples id list
      # illumina_bam_list.txt: Illumina bam file list
      

      • MELT version 2.2.2

      java -jar ./MELTv2.2.2/MELT.jar Single -h GCA_000001405.15_GRCh38_no_alt_analysis_set.fa -bamfile $bam_file -n ./MELTv2.2.2/add_bed_files/Hg38/Hg38.genes.bed -t ./MELTv2.2.2/me_refs/Hg38/mei_list.txt -w $result_dire
      

  • ONT

    • Single Cell

      • Palmesom

      See https://github.com/HelloYanming/PALMESOM

    • Bulk

      • Palmer version 2.0.0

      # L1
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type LINE --mode raw --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      # Alu
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type ALU --mode raw --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      # SVA
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type SVA --mode raw --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      

      • xTea_long version 0.1.0

      SAMPLE_ID=./sample_id.txt
      BAMS=./sample_bams.txt
      WFOLDER=$Work_dire
      OUT_SCRTP=$Work_dire/submit_jobs.sh
      XTEA=./xTea/bin/xtea/
      RMSK=./xTea/bin/xtea/xtea_consensus/LINE/hg38/hg38_L1_larger_500_with_all_L1HS.out
      CNS_L1=./xTea/bin/xtea/xtea_consensus/consensus/LINE1.fa
      REP_LIB=./xTea/bin/xtea/xtea_consensus/
      
      python ${XTEA}/xtea_long/gnrt_pipeline_local_long_read_v38.py  -i ${SAMPLE_ID} -b ${BAMS} -p ${WFOLDER} -o ${OUT_SCRTP} --xtea ${XTEA} -n 16 -m 48 -t ${TIME} --rmsk ${RMSK} --cns ${CNS_L1} --rep ${REP_LIB}  --min 4000  -f 31 -y 15 --clean --ref GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      

  • TEnCATS

  TBD

  • Assembly Contig

    • Palmer version 2.0.0

      # L1
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type LINE --mode asm --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      # Alu
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type ALU --mode asm --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      # SVA
      ./PALMER --input $bam_file --workdir $work_dire --ref_ver GRCh38 --output $output --type SVA --mode asm --chr $chr --ref_fa GCA_000001405.15_GRCh38_no_alt_analysis_set.fa
      
      

    • PAV version 2.3.4

      singularity run --bind $pwd:$pwd library://becklab/pav/pav:latest -c 16
      
      # Under the same folder, we included config.json which contains the reference file path and assemblies.tsv which contains the name and path of our phased assembly contigs. The reference we used here is GCA_000001405.15_GRCh38_no_alt_analysis_set.fa. 
      

• SNV

  • Illumina

    • Bulk

      • GATK Mutect2 version 4.3.0.0

      gatk Mutect2 \
      -I $bam_file \
      -R GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      -O $mutect2_output_file
      

      • MosaicForecast version 0.0.1

      Clean mutect2 output to get potential coordinates:

      singularity exec mosaicforecast_0.0.1.sif \
      python ./MosaicForecast/MuTect2-PoN_filter.py \
      $illumina_bulk.bed \
      ./mutect2_output_file \
      ./MosaicForecast/resources/SegDup_and_clustered.GRCh38.bed
      

      ReadLevel features extraction:

      singularity exec mosaicforecast_0.0.1.sif \
      python ./MosaicForecast/ReadLevel_Features_extraction.py \
      ./$illumina_bulk.bed \
      ./$illumina_bulk.features \
      $bam_file \
      ./GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      ./MosaicForecast/hg38/k24.umap.wg.bw \
      16 \
      bam
      

      Genotyping:

      singularity exec mosaicforecast_0.0.1.sif \
      Rscript ./MosaicForecast/Prediction.R \
      ./$illumina_bulk.features \
      ./MosaicForecast/models_trained/50xRFmodel_addRMSK_Refine.rds Refine \
      ./$bulk.genotype
      

      • DeepVariant version 1.6.0

      singularity exec deepvariant_1.6.0.sif \
      /opt/deepvariant/bin/run_deepvariant \
      --model_type=WGS \
      --ref=./GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --reads=$bam_file \
      --output_vcf=$out.vcf \
      --num_shards=12
      

      • Clair3 version 1.0.6

      singularity exec clair3_latest.sif \
      /opt/bin/run_clair3.sh \
      --bam_fn=$bam_file \
      --ref_fn=GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --threads=18 \
      --platform="ilmn" \
      --model_path=/opt/models/ilmn \
      --output=$output_dire
      

      • ClairS-TO version 0.0.2

      singularity exec clairs-to_latest.sif \
      /opt/bin/run_clairs_to \
      --tumor_bam_fn $bam_file \
      --ref_fn ./GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --threads 18 \
      --platform "ilmn" \
      --output_dir $output_dire \
      -conda_prefix /opt/micromamba/envs/clairs-to
      

    • Single Cell

    We used samtools mpileup to check if the AF presence in single cell bam files.

    samtools mpileup -q 15 -d 90 -f GCA_000001405.15_GRCh38_no_alt_analysis_set.fa --positions $Potential_coordinates --output-extra MAPQ $bam_file
    
    # And the output is processed by TODO
    
    

  • ONT

    • Bulk

      • DeepVariant version 1.6.0

      singularity exec deepvariant_1.6.0.sif \
      /opt/deepvariant/bin/run_deepvariant \
      --model_type=ONT_R104 \
      --ref=/GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --reads=$bam_file \
      --output_vcf=$out.vcf \
      --num_shards=36
      

      • Clair3 version 1.0.6

      singularity exec clair3_latest.sif \
      /opt/bin/run_clair3.sh \
      --bam_fn=$bam_file \
      --ref_fn=GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --threads=18 \
      --platform="ont" \
      --model_path=./rerio/clair3_models/r1041_e82_400bps_sup_v410 \ #downloaded from https://github.com/nanoporetech/rerio
      --output=$out_dire
      

      • ClairS-TO version 0.0.2

      singularity exec ./clairs-to_latest.sif \
      /opt/bin/run_clairs_to \
      --tumor_bam_fn $bam_file \
      --ref_fn ./GCA_000001405.15_GRCh38_no_alt_analysis_set.fa \
      --threads 18 \
      --platform ont_r10_dorado_sup_4khz \
      --output_dir $output_dire \
      --conda_prefix /opt/micromamba/envs/clairs-to
      

    • Single Cell

    We used samtools mpileup to check if the AF presence in single cell bam files.

    samtools mpileup -q 15 -d 90 -f GCA_000001405.15_GRCh38_no_alt_analysis_set.fa --positions $Potential_coordinates --output-extra MAPQ $bam_file
    
    # And the output is processed by TODO
    
    

  • Assembly Contig

    • PAV version 2.3.4

      singularity run --bind $pwd:$pwd library://becklab/pav/pav:latest -c 16
      
      # Under the same folder, we included config.json which contains the reference file path and assemblies.tsv which contains the name and path of our phased assembly contigs. The reference we used here is GCA_000001405.15_GRCh38_no_alt_analysis_set.fa. 
      

Data Analysis