Updated: Sep 19, 2023

Biastools: Measuring, visualizing and diagnosing reference bias

This github is originally forked from https://github.com/sheila12345/biastools

Prerequisite programs:

• samtools=v1.11
• bcftools=v1.9
• bedtools=v2.30.0
• gzip=v1.9
• tabix=v1.9
• bowtie2=v2.4.2
• bwa=v0.7.17
• mason_simulator=v2.0.9
• SeqAn=v2.4.0

Install

• pip

pip install biastools

• Github

git clone https://github.com/maojanlin/biastools.git
cd biastools

Though optional, it is a good practice to install a virtual environment to manage the dependancies:

python -m venv venv
source venv/bin/activate

Now a virtual environment (named venv) is activated:

python setup.py install

Usage

Simulation, plotting, and analysis

$ biastools --simulate --align --analyze -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_id>

With the example command, biastools

1. Simulates reads based on <ref.fa> and <vcf>, generating pair-end .fq.gz files for both haplotypes (work_dir/sample_name.hap{A,B}_{1,2}.fq.gz).
2. Aligns the reads to the reference <ref.fa>, generating a BAM file with phasing information (work_dir/sample_name.run_id.sorted.bam).
3. Analyzes the BAM file with the context-aware assignment method, generating bias reports and plots.

Other aligners

Biastools supports Bowtie 2 and bwa mem aligners. BAM files from other aligners (named with <work_dir/sample_name.run_id.sorted.bam> and tagged with haplotype information) can be analyzed with

$ biastools --analyze -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_id>

Real sequence data

Biastools can also analyze real sequence data with the -R option using the context-aware assignment algorithm. The resulting plot does not include simulation information (sample_id.real.indel_balance.pdf).

$ biastools --analyze -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_id> -R

Multiple indel plots

Multiple analysis result can be combined into one single indel-balance plot.

$ biastools --analyze -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_id> \
                      -lr file1.bias file2.bias file3.bias... \
                      -ld run_id1 run_id2 run_id3...

The output file sample_name.combine.sim.indel_balance.pdf merges the indel information of the bias reports after the -l option with the simulated balance information. For real bias report only, using the -R option to generate combined plot file sample_name.combine.real.indel_balance.pdf without simulated balance.

Bias prediction from bias report

Real data

Biastools can predict if a variant is bias or not by:

$ biastools --predict -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_pd_id> -pr <path_to_bias_report>

With the example command, biastools 4. Generates two files: sample_name.real.pd_id_bias.tsv and sample_name.real.pd_id_suspicious.tsv. The bias.tsv report contains all sites predicted to be biased by the model. The suspicious.tsv file contains the sites which suspicious of lacking enough information from the VCF file. In another word, the reads align to the site shows different pattern to the haplotype indicated by the VCF file.

Simulated guided prediction

$ biastools --predict -o <work_dir> -g <ref.fa> -v <vcf> -s <sample_name> -r <run_pd_id> \
                      -pr <path_to_bias_report> \
                      -ps <path_to_simulated_bias_report>

If the report of the sample based on simulated data is presented, biastools can generate cross prediction experiment result. In the experiment, the ground truth bias sites are based on simulation data.

Scanning bias without vcf information

Scanning

$ biastools_scan --scan -o <work_dir> -g <ref.fa> -s <sample_name> -r <run_id> -i <path_to_target.bam>

Biastools transforms the <path_to_target.bam> into the mpileup format and generates baised and suspicious regions (sample_name.run_id.bias.bed and sample_name.run_id.suspicious.bed).

Compare two bam files with common baseline

$ biastools_scan --compare_bam -o <work_dir> -g <ref.fa> -s <sample_name> -r <run_id> \
                               -i  <path_to_target.bam> \
                               -i2 <path_to_second.bam> \
                               -m  <path_to_target.mpileup> \
                               -m2 <path_to_second.mpileup>

Biastools generates a common baseline from path_to_target.bam and path_to_second.bam, and uses the new common baseline to recalculate the bias regions based on the two mpileup files. The mpileup files can be generated by running scanning first, or directly run the bcftools consensus.

Directly compare two bias reports

User can also generate the comparison of the bias reports without a common baseline (not recommended):

$ biastools_scan --compare_rpt -o <work_dir> -s <sample_name> -r <run_id> \
                               -b1 <path_to_target_bias.bed> \
                               -b2 <path_to_improved_bias.bed> \
                               -l2 <path_to_improved_lowRd.bed>