SpatialSNV

A novel method for calling and analyzing SNVs from spatial transcriptomics data.

We divided the process of calling mutations from spatial transcriptomics data into two parts: Data Preprocessing and Data Analysis.

All analyzed jupyter notebooks are saved in the article folder

Install

To install spatialsnv, use pip:

pip install spatialsnv

We recommend using Python version 3.10.14. You also need to install the following tools: samtools,gatk,picard

Data Preprocessing

Splitting BAM File by Chromosome for Speed Up (Optional)

spatialsnvtools SplitChromBAM -b demo.bam –s demo –o demo_split -@ 10 --only_autosome

Options:

• -b, --bam FILE
  BAM file that needs to be split by chromosome [required]
• -s, --sample TEXT
  Sample ID [required]
• -o, --outdir TEXT
  Output directory for the split BAM files [required]
• -@, --threads INTEGER
  Sets the number of threads
• --only_autosome
  Only analyze autosomes
• --help
  Show this message and exit.

Mutation Calling Data Preprocessing

10x or drop-seq

spatialsnvtools PerpareBAMforCalling \
    -b demo.bam \
    -o process_out \
    -s demo \
    -c 'CR' \
    -u 'UR' \
    -@ 10 \
    --fasta GRCh38.p12.genome.fa \
    --dbsnp dbsnp.chr9.hg38.vcf.gz \
    --removetmp \
    --picard $pathtopicard/picard.jar \
    --gatk $pathtogatk/gatk \
    --samtools $pathtosamtools/samtools

stereo-seq

spatialsnvtools PerpareBAMforCalling \
    -b demo.stereo.bam \
    -o stereo_process_out \
    -s stereo_demo \
    --stereo \
    --gem demo.gem.gz \
    -x 0 -y 0 -@ 10 \
    --fasta GRCh38.p12.genome.fa \
    --dbsnp dbsnp.chr9.hg38.vcf.gz \
    --removetmp \
    --picard $pathtopicard/picard.jar \
    --gatk $pathtogatk/gatk \
    --samtools $pathtosamtools/samtools

Options:

• -b, --bam FILE
  BAM file that needs preprocessing [required]
• -o, --outdir TEXT
  Output directory for the preprocessing results [required]
• -s, --proxy TEXT
  Sample ID [required]
• -f, --fasta FILE
  Reference FASTA used for mutation calling [required]
• -d, --dbsnp FILE
  dbSNP file used for BQSR [required]
• -c, --barcode TEXT
  Cell Barcode in BAM file (e.g., CR for 10X Genomics)
• -u, --umi TEXT
  UMI (Molecular Barcodes) in BAM file (e.g., UR for 10X Genomics)
• --stereo
  Ensure that your data is stereo (barcode is Cx and Cy)
• --gem TEXT
  GEM file matching your raw stereo BAM
• -x, --xsetoff INTEGER
gem_x + x_offset = bam_x
• -y, --ysetoff INTEGER
gem_y + y_offset = bam_y
• --tmpdir TEXT
  Specify a temporary directory
• -@, --threads INTEGER
  Sets the number of threads
• --samtools TEXT
  Specify the path to samtools, if not specified, automatically detected
• --picard TEXT
  Specify the path to picard.jar, if not specified, automatically detected
• --gatk TEXT
  Specify the path to GATK, if not specified, automatically detected
• --removetmp
  Remove all temporary files
• --help
  Show this message and exit.

SNV Calling on Preprocessed BAM Files

spatialsnvtools SNVCalling \
    -b demo.processed.bam \
    -s demo \
    -o demo.vcf.gz \
    -f GRCh38.p12.genome.fa \
    --pon 1000g_pon.hg38.vcf.gz \
    --germline af-only-gnomad.hg38.vcf.gz

Options:

• -s, --sample TEXT
  Sample ID (e.g., -b example.bam1 -b example.bam2) [required]
• -b, --bam FILE
  BAM file(s) with index (e.g., -b example.bam1 -b example.bam2) [required]
• -o, --outvcf TEXT
  Output VCF file [required]
• -f, --fasta FILE
  Reference FASTA file [required]
• --pon FILE
  Panel of Normals (PON) file
• --germline FILE
  Germline source file
• --gatk TEXT
  Specify the path to GATK
• -L, --chrom TEXT
  Specify chromosome(s) to analyze
• --help
  Show this message and exit.

Traceback SNVs to Spatial Transcriptomics

10x or drop-seq

spatialsnvtools CallBack \
    --bam demo.processed.bam \
    --vcf demo.vcf.gz \
    -o demo_matrix \
    -s demo \
    --tmpdir demo_tmp \
    --only_autosome \
    -c "CB"  \
    -u "UB" \
    -@ 1

stereo-seq

spatialsnvtools CallBack\
    --bam demo.stereo.bam \
    --vcf demo.stereo.vcf.gz \
    -o demo_stereo_matrix \
    -s demo_stereo \
    --tmpdir demo_stereo_tmp \
    --stereo \
    -x 0 -y 0 --binsize 100 \
    --only_autosome \
    -@ 1 \
    --umi UM \
    --removetmp

Options:

• -b, --bam FILE
  BAM file(s) with index (e.g., -b example.bam1 -b example.bam2) [required]
• -v, --vcf FILE
  VCF file for SNV data [required]
• -s, --sample TEXT
  Sample ID [required]
• -o, --outdir TEXT
  Output directory [required]
• --tmpdir TEXT
  Specify a temporary directory
• --only_autosome
  Only analyze autosomes
• --stereo
  Ensure that your data is stereo (barcode is Cx and Cy)
• -c, --barcode TEXT
  Cell Barcode in BAM file (e.g., CR for 10X Genomics)
• -u, --umi TEXT
  UMI (Molecular Barcodes) in BAM file (e.g., UR for 10X Genomics)
• -@, --threads INTEGER
  Sets the number of threads
• --qmap INTEGER
  Sets the number of qmap
• -x, --xsetoff INTEGER
gem_x + x_offset = bam_x
• -y, --ysetoff INTEGER
gem_y + y_offset = bam_y
• --binsize INTEGER
  Set the bin size
• --removetmp
  Remove all temporary files
• --help
  Show this message and exit.

All germline resource data (hg38) is stored in the GigaDB dataset.

Data Analysis

Please refer to the data_process_snv.ipynb file in the demo folder for the basic operations of spatialSNV.

Contact

For inquiries or support, please contact:
Young: [liyang13@genomics.cn] Yi: [liuyi6@genomics.cn]