WASP2: Allele-specific pipeline for unbiased read mapping and allelic-imbalance analysis

Requirements

• Python >= 3.7
• numpy
• pandas
• polars
• scipy
• pysam
• pybedtools
• typer
• anndata

Installation

Recommended installation through conda, and given environment

conda env create -f environment.yml

 

Allelic Imbalance Analysis

Analysis pipeline currently consists of two tools (Count and Analysis)

 

Count Tool

Process allele specific read counts per SNP.
Sample names can be provided in order to filter out non-heterozygous SNPs. Genes and ATAC-seq peaks can also be provided to include SNPs that overlap regions of interest.
Providing samples and regions is highly recommended for allelic-imbalance analysis

Usage

python WASP2/src/counting count-variants [BAM] [VCF] {OPTIONS}

Required Arguments

• BAM file containing aligned reads.
• VCF file containing SNP info

Optional Arguments

• -s/--samples: Filter SNPs whose genotypes are heterozygous in one or more samples. Accepts comma delimited string, or file with one sample per line.
• -r/--region: Filter SNPs that overlap peaks/regions of interest. Accepts files in narrowPeak, BED, gtf and gff3 format.
• -o/--out_file: Output file for counts. Defaults to counts.tsv
• -t/--temp_loc: Write intermediary files to a directory instead of deleting. Useful for debugging issues.
• --use_region_names: If regions are provided use region names as identifiers instead of coordinates. Names are denoted in fourth column of BED. Ignored if no name column in BED file.

RNA-Seq Specific Arguments

• --gene_feature: Feature type in gtf/gff3 for counting intersecting SNPs. Defaults to 'exon' for snp counting.
• --gene_attribute: Attribute name from gtf/gff3 attribute column to use as ID. Defaults to '_id' in gtf and 'ID' in gff3.
• --gene_parent: Parent attribute in gtf/gff3 for feature used in counting. Defaults to 'transcript_id' in gtf and 'Parent' in gff3.

 

Analysis Tool

Analyzes Allelic Imbalance per ATAC peak given allelic count data

Usage

python WASP2/src/analysis find-imbalance [COUNTS] {OPTIONS}

Required Arguments

• COUNTS: Output data from count tool

Optional Arguments

• -o/--out_file: Output file to write analysis results to. (Default. ai_results.tsv)
• --min: Minimum allele count needed for analysis. (Default. 10)
• -p/--pseudocount: Pseudocount added when measuring allelic imbalance. (Default. 1)
• --phased: Calculate allelic imbalance using phased haplotype model. By default, calculates AI assuming unphased/equal likelihood for each haplotype.
• --region_col: Name of region column for current data. Use 'region' for ATAC-seq. Plans for 'genes' for RNA-seq and 'SNP' for per SNP. Recommended to leave blank. (Default: Auto-parses if none provided)
• --groupby: Report allelic imbalance by parent group instead of feature level in RNA-seq counts. Name of parent column. Not valid if no parent column or if using ATAC-seq peaks. (Default: Report by feature level instead of parent level)

 

Unbiased Allele-Specific Read Mapping

Mappability filtering pipeline for correcting allelic mapping biases.
First, reads are mapped normally using a mapper chosen by the user (output as BAM). Then mapped reads that overlap single nucleotide polymorphisms (SNPs) are identified. For each read that overlaps a SNP, its genotype is swapped with that of the other allele and the read is re-mapped. Re-mapped reads that fail to map to exactly the same location in the genome are discarded.

Step 1: Create Reads for Remapping

This step identifies reads that overlap snps and creates reads with swapped alleles.

Usage

python WASP2/src/mapping make-reads [BAM] [VCF] {OPTIONS}

Required Arguments

• BAM file containing aligned reads.
• VCF file containing SNP info

Optional Arguments

• --threads: Threads to allocate.
• -s/--samples: Filter Polymorphic SNPs in one or more samples. Accepts comma delimited string, or file with one sample per line.
• -o/--out_dir: Output directory for data to be remapped
• -t/--temp_loc: Write intermediary files to a directory instead of deleting. Useful for debugging issues.
• -j/--out_json: Output json containing wasp file info to this file instead of default. Defaults to [BAM_PREFIX]_wasp_data_files.json

Step 2: Remap Reads

Remap fastq reads using mapping software of choice

Example

bwa mem -M "BWAIndex/genome.fa" "${prefix}_swapped_alleles_r1.fq" "${prefix}_swapped_alleles_r2.fq" | samtools view -S -b -h -F 4 - > "${prefix}_remapped.bam"
samtools sort -o "${prefix}_remapped.bam" "${prefix}_remapped.bam"
samtools index "${prefix}_remapped.bam"

Step 3: Filter Reads that Fail to Remap

Identify and remove reads that failed to remap to the same position. Creates allelic-unbiased bam file

Usage

python WASP2/src/mapping filter-remapped "${prefix}_remapped.bam" --json "${prefix}_wasp_data_files.json"

OR

python WASP2/src/mapping filter-remapped "${prefix}_remapped.bam" "${prefix}_to_remap.bam" "${prefix}_keep.bam"

Required Arguments

• Remapped BAM File
• Either: json or to_remap_bam + keep.bam
  • -j/--json: json containing wasp file info. Default output from make-reads: [BAM_PREFIX]_wasp_data_files.json
  • to_remap_bam: to_remap_bam used to generate swapped alleles. Default: [BAM_PREFIX]_to_remap.bam
  • keep_bam: BAM containing reads that were not remapped. Default: [BAM_PREFIX]_keep.bam

Optional Arguments

• --threads: Threads to allocate.
• -o/--out_bam: File to write filtered bam. Defaults to [BAM_PREFIX]_wasp_filt.bam.
• --remap_keep_bam: Output bam file with kept reads to this file if provided.
• --remap_keep_file: Output txt file with kept reads names to this file if provided.

 

Single-Cell Allelic Counts

Process allele specific read counts for single-cell datasets.
Output counts as anndata containing cell x SNP count matrix.

Usage

python WASP2/src/counting count-variants-sc [BAM] [VCF] [BARCODES] {OPTIONS}

Required Arguments

• BAM file containing aligned reads.
• VCF file containing SNP info
• BARCODE file used as index, contains one cell barcode per line

Optional Arguments

• -s/--samples: Filter SNPs whose genotypes are heterozygous in one or more samples. Accepts comma delimited string, or file with one sample per line. RECOMMENDED TO USE ONE SAMPLE AT A TIME.
• -f/--feature: Features used in single-cell experiment. Filter SNPs that overlap regions/features of interest. Accepts BED formatted files.
• -o/--out_file: Output file for counts. Defaults to allele_counts.h5ad
• -t/--temp_loc: Write intermediary files to a directory instead of deleting. Useful for debugging issues.

 

Single-Cell Allelic Imbalance

Estimate allele-specific chromatin acccessibility using single-cell allelic counts.
Allelic-Imbalance is estimated on a per-celltype basis.

Usage

python WASP2/src/counting find-imbalance-sc [COUNTS] [BARCODE_MAP] {OPTIONS}

Required Arguments

• COUNTS file (.h5ad) containing matrix of single-cell allelic counts.
• BARCODE MAP: Two column TSV file mapping specific cell barcodes to some group/celltype.
  Each line following format ... [BARCODE] \t [CELLTYPE]

Optional Arguments

• -o/--out_file: Output file to write analysis results to. (Default. ai_results_[GROUP].tsv)
• --min: Minimum allele count needed for analysis. (Default. 10)
• -p/--pseudocount: Pseudocount added when measuring allelic imbalance. (Default. 1)
• -s/--sample: Use het genotypes for this sample in count matrix. Automatically parse if data contains 0 or 1 sample. REQUIRED IF MULTIPLE SAMPLES IN DATA.
• --phased: Calculate allelic imbalance using phased haplotype model. By default, calculates AI assuming unphased/equal likelihood for each haplotype.
• --unphased: Explicitly use unphased model.
• -z/--z_cutoff: Remove SNPS and associated regions whose counts exceed Z-score cutoff. Extra layer of QC for single-cell allelic counts

 

Single-Cell Comparative Imbalance

Compare differential allelic-imbalance between celltypes/groups.

Usage

python WASP2/src/counting compare-imbalance [COUNTS] [BARCODE_MAP] {OPTIONS}

Required Arguments

• COUNTS file (.h5ad) containing matrix of single-cell allelic counts.
• BARCODE MAP: Two column TSV file mapping specific cell barcodes to some group/celltype.
  Each line following format ... [BARCODE] \t [CELLTYPE]

Optional Arguments

• -o/--out_file: Output file to write analysis results to. (Default. ai_results_[GROUP1]_[GROUP2].tsv)
• --groups/--celltypes: Specific groups in barcode map to compare differential allelic imbalance. If providing input requires 2 groups minimum, otherwise compare all group combinations.
• --min: Minimum allele count needed for analysis. (Default. 10)
• -p/--pseudocount: Pseudocount added when measuring allelic imbalance. (Default. 1)
• -s/--sample: Use het genotypes for this sample in count matrix. Automatically parse if data contains 0 or 1 sample. REQUIRED IF MULTIPLE SAMPLES IN DATA.
• --phased: Calculate allelic imbalance using phased haplotype model. By default, calculates AI assuming unphased/equal likelihood for each haplotype.
• --unphased: Explicitly use unphased model.
• -z/--z_cutoff: Remove SNPS and associated regions whose counts exceed Z-score cutoff. Extra layer of QC for single-cell allelic counts