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De-novo assembly graph experiments in yeast

This directory contains the snakemake pipelines and other scripts for SV genotyping using vg on 12 yeast strains. Primarily, the SV genotyping performance of two different vg graphs is compared:

  • VCF graph: Created with vg construct using S288C as reference strain and VCFs with SVs from Assemblytics (Nattestad et al.), AsmVar (Huang et al.) and paftools (Li) as variants
  • Cactus graph: Created from multiple genome alignment of yeast strains using cactus and hal2vg

The experiments comprise several phases which are explained in the following:

1. Prepare yeast assemblies and call SVs relative to the reference strain S288C

git clone https://github.com/vgteam/sv-genotyping-paper.git
cd yeast/assemblies
snakemake

This will clone this repo including the contained PacBio assemblies from the Yeast Population Reference Panel (YPRP). Subsequently, it will run 3 SV calling pipelines on the 11 non-reference strains:

Because the resulting callsets differ considerably, the union of all 3 callsets is produced (high-sensitivy callset). Finally, the high-sensitivity callsets of all 11 non-reference strains are merged.

2. Download Illumina reads for the 12 yeast strains

The reads mapped to the yeast graphs were downloaded from SRA: SRR4074413, SRR4074412, SRR4074411, SRR4074394, SRR4074385, SRR4074384, SRR4074383, SRR4074358, SRR4074258, SRR4074257, SRR4074256, SRR4074255.

cd yeast/illumina_reads
snakemake

3. Create, index and map Illumina reads to the VCF graph

There are 2 different subdirectories for the VCF graph in yeast/graphs:

  • yeast/graphs/constructunion_all - Integrate variants from all strains into graph
  • yeast/graphs/constructunion_five - Integrate variants from 3 S. cerevisiae and 2 S. paradoxus strains into the graph

Let's call them the two strain sets. For the first strain set (all), run:

cd yeast/graphs/constructunion_all
snakemake

For the second strain set (five), run:

cd yeast/graphs/constructunion_five
snakemake

This will create the VCF graphs for the two strain sets using S288C as reference strain and the high-sensitivity variant callsets produced before.

As a result yeast/graphs/constructunion_*/mappings will contain the sorted GAM alignments of the Illumina reads against the graph.

4. Run cactus to produce an assembly-derived graph

The cactus genome-genome aligner is run using toil. To install toil in a virtual environment:

pip install virtualenv
virtualenv cactus_env
source cactus_env/bin/activate
pip install --upgrade toil

Like for the VCF graph, there are 2 different strain sets for the cactus graph: all and five. To create cactus graphs for both of them, now follow the steps in yeast/cactus/all/aws_commands.sh and yeast/cactus/five/aws_commands.sh.

5. Create, index and map Illumina reads to the cactus graph

cd yeast/graphs/cactus_all
snakemake

cd yeast/graphs/cactus_five
snakemake

This will a) create each of the two cactus graphs from the cactus alignments produced in the previous step and b) map the Illumina reads to them.

As a result yeast/graphs/cactus_*/mappings will contain the sorted GAM alignments of the Illumina reads against the graphs. Additionally, it will produce png plots of mapping quality and identity in yeast/graphs/cactus_*/mappings/stats.

6. Genotype SVs on the VCF graph and the cactus graph

The SV genotyping is run using toil-vg. First, install toil in a separate virtual environment:

virtualenv toilvenv
source toilvenv/bin/activate
pip install toil[aws,mesos]==3.18.0
pip install toil-vg

For the VCF graph, follow the steps in yeast/vg_call/graphA_aws_commands.sh. It contains commands for the all strain set but can be easily modified for the five strain set. For the cactus graph, follow the steps in yeast/vg_call/graphB_aws_commands.sh. It contains commands for the all strain set but can be easily modified for the five strain set.

The compressed vcf results are written into an outstore on S3 of the form aws:us-west-2:vgcall-yeast-<graph>-<strain-set>-outstore

7. Evaluate genotyping performance

To evaluate the SV genotype calls made by vg call you need to download them from the S3 outstore to the respective local directory yeast/evaluation/calls/<graph>_<strain-set>/vcf. This can be done via the S3 web interface or commands of the form aws s3 cp s3://vgcall-yeast-<graph>-<strain-set>-outstore/<sample>*.vcf.gz yeast/evaluation/calls/<graph>_<strain-set>/vcf.

Now, the evaluation pipeline can be started with:

source toilvenv/bin/activate
cd yeast/evaluation/
snakemake

It will produce pdf plots showing average deltas in mapping identity and mapping quality of reads aligned to the sample graphs in yeast/evaluation/results/assemblyeval_svonly.