SpikeVar & TykeVar: Simulation of Mosaic Variants in Sequencing Data

Hackathon team: Lead: Fritz Sedlazeck - Developers: Xinchang Zheng, Michal Izydorczyk, Chi-Lam Poon, Philippe Sanio, Farhang Jaryani, Joyjit Daw, Divya Kalra, Adam Alexander - Writers: Erik Stricker, Sontosh Deb

We provide two simulation workflows which output sequencing read files with artificial mosaic variants and a ground truth mosaic variant annotation file for the validation of mosaic variant callers.

Table of Contents

1. Background 2. Installation 3. Dependencies 4. Tests 5. How to Use It 6. Example Implementation 7. Method Description 8. Contributers 9. References	Spike_and_Tyke.mp4

Background

In the context of individual genome comparison, mutations that appear within a small fraction of the population are considered rare variants¹. When assessing a population of cells from a tissue of the same individual in turn, rare variants only present in a small fraction of the cells are defined as mosaic variants (MVs)². Recent studies have shown that there is potential disease associations of for certain MVs². However, MVs are challenging to detect because they are mixed in with data from the non-mutated cells and present in the same sequencing file. Therefore, several pipelines have been developed or adjusted to extract mosaic single nucleotide, structural or indel variants from whole genome sequencing data such as Sniffles³, DeepMosaic⁴, Mutect2⁵, DeepVariant⁶. To benchmark and validate the efficiency and accuracy of these methods, sequencing files with known MVs are necessary. We developed two simulation workflows called SpikeVar (Spike in Known Exogenous Variants) and TykeVar (Track in Your Key Endogenous Variants), which output sequencing read files with artificial MVs and a ground truth annotation file for the MVs. SpikeVar accomplishes this by spiking in real reads from a sample at user-defined ratio into the sequencing file from a second sample. In contrast, TykeVar creates a list of random mutations and modifies a fraction of existing reads to match the user-defined MV frequency.

Installation

These instructions are valid for Linux x86. For other platforms (e.g. MacOS), instructions will need to be adapted.

Installing two conda environments with python 3.10

conda create -n mosaicSim python=3.10

conda activate mosaicSim

This tool is written fully in python. To install the relevant dependencies, run

pip install -r $REPO_ROOT/requirements.txt

where $REPO_ROOT is the root folder of the repository.

Once the requirements are installed, please install the following additional packages

conda install -c bioconda samtools bcftools mosdepth

Dependencies

SpikeVar

• mosdepth 0.3.2
• samtools 1.15.1
• Python 3.6.8
• bcftools

TykeVar

• pysam (0.21.0)
• numpy (1.25.2)
• biopython (1.81)

Tests

The repository comes with unit tests to ensure correct setup and functioning of core libraries.

To run the unit tests, please execute

python -m pytest tests/*

How to Use It

SpikeVar

The spiked-in dataset simulates a sample with potential mosiac variants at a user-specified ratio. The re-genotyped VCFs of the samples and the VCF of the spiked-in dataset can be compared to evaluate AF < user-specified value.

1) SpikeVarDatabaseCreator - Generate Spiked-in Dataset

In this step, x% of mutations are strategically introduced from sample A to sample B. Both datasets are down-sampled and then merged to create a mixed dataset that represents a sequence read dataset with mosaic variants, including structural variations (SVs), single nucleotide variations (SNVs), and insertions/deletions (indels).

./spike-in.sh <path to sampleA.bam> <path to sampleB.bam> <spike-in ratio x/100> <path to samtools binary> <path to mosdepth binary> <output dirpath> <path to script calculate_ratio.py>

2) SpikeVarReporter - Filter Reads After Variant Allele Frequency Recalculation

After creating the modified BAM file we have to re-calculate the variant allele frequency (VAF) for all variants. First, all variants stored from both VCF files must be merged and the VAF must be recalculated. Depending on the variants we either start a SNV or SV caller, which can recalculate the VAF of each variant. For SNVs, we are using bcftools mpileup. For SVs and short read data we are using Paragraph from Illumina and for long read data Sniffles2 is used.

Last the re-genotyped VCF is filtered according to the VAF with a small Python script, which calculates the minor allele frequency (MAF) for each variant and lets a variant pass to the final output if the MAF is equal or greater than the use specified VAF.

./2b_re-genotyping_main.sh <VARIANT> <VAF> <sampleA.vcf> <sampleB.vcf> <sampleAandB.bam> output/path <ref.fa> <short|long>

3) Run Your Favorite Mosaic Variant Caller and Compare Results

TykeVar

Once the Python dependencies are installed, the scripts can be run directly from the scripts/Tyke subfolder.

1) TykeVarSimulator - Generate Simulated VCF

The VCF simulator generates a random set of mosaic variants (SNVs and SVs). The variants can be parameterized with VAF, the number of variants to simulate, and the size of the variations. The generated file is in the Sniffles VCF format.

The variants generated here act as an input into the read editor step (described below) which produces modified reads with the variants inserted into them. The same VCF file is also the ground truth for evaluating mosaic variant callers.

python vcfgen.py <path_to_bam> <path_to_ref> <output_path_prefix>

2) TykeVarEditor - Generate Edited Reads Based on Simulated VCF

python main.py -v <SIMULATED_VCF> -b <BAM> -r <REF> -o <OUTPUT_FASTQ>

This command above takes in the VCF which determines which variants to introduce into the reads. The BAM file is used to find the reads which overlap with variant locations. Only a subset of the reads corresponding to a particular variant location are edited. This is determined by the allele frequency. The output FASTQ file has the edited reads. The query name of each read is kept the same.

3) TykeVarMerger - Re-Align Modified Reads and Merge Them

Replace the reads into the original dataset with the modified reads.

Note that before running the merge and filter step, the modified reads need to be mapped against the original reference.

python filter_merge_bam.py -b <BAM> -m <MOD_BAM> --primary -o <OUT_DIR> --prefix <PREFIX>

In the above cmd:

• BAM is the original dataset.
• MOD_BAM has the alignments generated by mapping the modified reads against the reference.
• --primary ensures that only primary alignments are merged back into the dataset.
• OUT_DIR is the output location for the merged and filtered final BAM.
• PREFIX is used to define a filename prefix for the output file.

4) Run Your Favorite Mosaic Variant Caller and Compare Results

For SV calling, we tried Sniffles:

sniffles --input <MOD_MERGED_BAM> --vcf sniffles_out.vcf --mosaic --threads 8

• MOD_MERGED_BAM is the output bam file from filter_merge_bam.py

for SNV calling, we ran longshot:

longshot --bam <MOD_MERGED_BAM> --ref <REF> --out longshot_out.vcf --min_cov 3

Example Implementation

SpikeVar

Here, we use the SpikeVar workflow to automatically spike in sample HG002 at a 5% concentration into sample HG0733, to result in a 5% mosaic variant allele frequency (VAF). A downside is that the generated mixed .bam file will include 4 haplotype structures which cannot be corrected for. Furthermore, certain variants (e.g. HG002 variants) will not be presented at the targeted VAF. For example, heterozygous variants will not be represented by 5% VAF but rather at ~2.5% VAF. To account for this we re-genotype variants and report only variants that should be identifiable at the user-defined threshold or higher VAF.

1) Fetch Data

In order to spike in sample B into sample A, the pipeline first needs an initial set of aligned reads. We used HG002 and HG00733 datasets.

Reads - ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/HG002_NA24385_son/Ultralong_OxfordNanopore/guppy-V3.2.4_2020-01-22/HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam and ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/HG002_NA24385_son/Ultralong_OxfordNanopore/guppy-V3.2.4_2020-01-22/HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam.bai

2) SpikeVarDatabaseCreator - Generate Spike-in Dataset

We spiked 5% reads from HG0733 to HG002 for the next part of the workflow.

./spike-in.sh HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam HG007733.bam 0.05 /software/bin/samtools /software/bin/mosdepth /output `pwd`/calculate_ratio.py

3) SpikeVarReporter - Filter Reads with >5% Variant Allele Frequency After Recalculation

After creating the new BAM file from e.g. HG002 and HG00733, we have to re-calculate the variant allele frequency (VAF) for all variants. First we merge both VCF files from e.g. HG002 and HG00733 with bcftools. Depending on the variants we either start a SNV or SV caller, which can recalculate the VAF of each variant. For SNVs we are using bcftools mpileup. For SVs and short read data we are using Paragraph from Illumina and for long read data Sniffles2 is used.

./2b_re-genotyping_main.sh SV 0.05 HG002_SV.Tier1.vcf HG00733_SV.Tier1.vcf /output/SPIKED.BAM ./ LONG hs37d5.fa

4) Run Your Favorite Mosaic Variant Caller

Run your choice of mosaic variant caller on the modified HG002_ONT_hg37_chr5_HG00733_ONT_hg37_chr5_merged.sorted.bam file and compare the results with the truth set output_genotypes_filtered.vcf file.

5) Results

Your spiked in reads are now visible in the IGV genome browser.

Example f a spiked in deletion.

Example of a spiked in inserton.

TykeVar

Here, we use the TykeVar workflow to modify reads of HG002 directly at their reference position by including artificial mutations. To demonstrate the wide application of this tool, we generate a random distribution of allele frequencies between 1% and 40%. In contrast to the above approach, we do not introduce new haplotypes with this. However, more complex mutations (e.g. rearrangements, duplication, or very long structural variants) will not be able to be introduced to the data itself, since the size of the reads is limited.

1) Fetch Data

In order to simulate and edit reads, the pipeline first needs an initial set of aligned reads and a reference. For our demonstration, we will use the GIAB datasets.

Reads - ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/HG002_NA24385_son/Ultralong_OxfordNanopore/guppy-V3.2.4_2020-01-22/HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam and ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/AshkenazimTrio/HG002_NA24385_son/Ultralong_OxfordNanopore/guppy-V3.2.4_2020-01-22/HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam.bai

Reference - https://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/release/references/GRCh37/hs37d5.fa.gz

We run the demonstration on chr22 only, so the dataset is filtered using

samtools view HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam 22 > chr22.HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam

2) TykeVarSimulator - Generate Variants and Modified Reads

First, we decompress the FASTA file.

gunzip hs37d5.fa.gz -c hs37d5.fa

Then we simulate variants

python vcfgen.py chr22.HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam hs37d5.fa chr22

3) TykeVarEditor - Add Modified Reads Back In

Generate a set of modified reads with inserted variants.

python main.py -v chr22SV.vcf -b chr22.HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam -r hs37d5.fa -o chr22.fastq 

4) TykeVarMerger - Re-Align Modified Reads and Merge Them

Once the new reads are generated, they need to be re-aligned and re-inserted back into the dataset by replacing the original reads.

We use minimap2 for both short-read and long-read alignment. In the example, we tested on chromosome 22.

# short read
minimap2 -ax sr hs37d5.fa.gz -t 14 --secondary=no chr22.fastq | \
    samtools view -bS --no-PG - > modified.chr22.bam

# long read
minimap2 -ax map-ont hs37d5.fa.gz -t 14 --secondary=no chr22.fastq | \
    samtools view -bS --no-PG - > modified.chr22.bam

Now that we have the alignments for modified reads, we then remove the old alignments with the same IDs of modified reads in the original bam, then insert the new alignments of modified reads back into this bam.

python filter_merge_bam.py -b chr22.HG002_hs37d5_ONT-UL_GIAB_20200122.phased.bam -m modified.chr22.bam

5) Run Your Favorite Mosaic Variant Caller

Run you choice of mosaic variant caller on the modified mod_chr22.bam file and compare the results with the simulated chr22SV.vcf file.

6) Results

The mod_chr22.bam file was run through Sniffles to determine potential mosaic variants. Then the ground truth VCF (chr22SV.vcf) and the VCF from Sniffles were both visualized on IGV to get a subjective view of whether the modified reads led to mosaic variants being introduced and detected. Below are 2 of several variants that overlapped between the ground truth and caller VCF and were confirmed in the underlying data.

A mosaic SV insertion.

A mosaic SV deletion.

A mosaic SNP.

Method Description

1. SpikeVar - Generation of Sequencing Data With a Low Frequencing of Reads From Another Sample

SpikeVar workflow, with major steps to assess the sensitivity and accuracy of the mosaic variant callers. (A, B: individual samples, A/B: merged samples, .bam and .vcf: input and output file formats in different steps, Black header boxes: tool or file names, Green header boxes: simulated files or final files used for validation comparisons)

The SpikeVar workflow outputs a mixed sequencing read dataset in .bam format containing reads from one dominant sample and reads from another sample spiked in at a user-defined ratio corresponding to the simulated mosaic variant allele frequency (VAF) together with a .vcf file annotating the confirmed mosaic variant locations within the mixed dataset. The SpikeVarDatasetCreator takes aligned sequencing reads from sample A and sample B as the initial input. In this step, a spike-in methodology is applied to strategically introduce x% of mutations from one sample to another using . Accordingly, sample A is first down-sampled to retain 100-x% of its original reads, then sample B is down-sampled to x% considering the coverage differences between the samples. Using , both down-sampled datasets are then merged to create a mixed dataset that represents a sequence read dataset with mosaic variants, including structural variations (SVs), single nucleotide variations (SNVs), and insertions/deletions (indels).

The SpikeVarReporter then determines VAFs for each variant in the mixed dataset using based on the mixed variant locations derived by merging the .vcf files from sample A and sample B using . Variants with VAFs exceeding or equal to the introduced mutations (i.e., x%) are then selected to create a truth set for benchmarking using .

To assess a mosaic variant caller’s sensitivity and accuracy, the same mixed dataset is used to call mosaic variants. The output mosaic variant locations and VAFs are then compared to the truth set for validation.

2. TykeVar - Creation of Sequencing Data With a Subset of Modified Reads

TykeVar workflow, with major steps to assess the sensitivity and accuracy of the mosaic variant callers. (A, B: individual samples, A/B: merged samples, .bam and .vcf: input and output file formats in different steps, Black header boxes: tool or file names, Green header boxes: simulated files or final files used for validation comparisons)

The TykeVar workflow produces a modified aligned sequence file in .bam format. This file contains modified reads simulating randomly positioned mosaic variants with user-defined VAF in random locations and is accompanied by a .vcf file containing the locations of the simulated mosaic variants with user-defined VAF.

The TykeVar workflow can be broadly split into 3 parts:

1. The TykeVarSimulator takes an aligned BAM, a reference, and several parameters such as range of VAF, variant sizes, etc. to generate a set of simulated mosaic SV and SNVs. It does so by choosing a random location and VAF from the given range and then evaluating whether that location has sufficient coverage for the desired VAF. If that condition is met, that variant is added to the output VCF.
2. The TykeVarEditor is responsible for inserting the simulated variants into the query sequences from the original dataset to generate modified reads with the mosaic variants built-in. The TykeVarEditor accepts a BAM, reference and the simulated VCF file as an input. Then for each variant, it fetches the overlapping reads from the BAM file, subsamples the reads to get the coverage that satisfies the desired VAF, and traverses the cigar string, query and reference sequences for each alignment to find the exact location to insert the variant. Once a modified read is created, it is written out into a FASTQ file. Note that for all new bases (SNVs or inserts), a q-score of 60 is chosen. The parsing and traversing of VCF, BAM and reference files are performed using APIs from pysam, biopython.SeqIO and numpy.
3. The TykeVarMerger re-introduces the modified reads into the original dataset. It does so by first removing the modified read ids from the input BAM to create a filtered BAM. Then the modified reads are aligned against the reference, and merged with the filtered BAM. The end result is a BAM with the same set of read ids as the original dataset, except with some reads modified to contain the mosaic variants. The output of this pipeline is thus a modified BAM and a VCF file which provides the truth set for the mosaic variants.

Contributers

Erik Stricker	Chi-Lam Poon	Philippe Sanio	Xinchang Zheng	Farhang Jaryani

Joyjit Daw	Michal Izydorczyk	Sontosh Deb	Fritz Sedlazeck	Adam Alexander

Divya Kalra

References

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