Sex Chromosome Identification by Negating Kmer Densities (SCINKD) is a wrapper to implicate the sex chromosome linkage group of a haplotype-resolved genome of the heterogametic sex with an unknown sex chromosome system. SCINKD [v2.0] is a Snakemake implementation (and additional outputting) of scinkd (v0.2).
At its core, SCINKD is a theoretical framework to identify sex chromosomes that operates under a few generalized assumptions of a diploid genome.
- Polymorphisms are broadly uniform between haplotypes within a single diploid individual.
- The density of genetic differences occur at much higher densities on the sex-limited region of the sex chromosomes
- This density is then identifiable by isolating haplotype-specific kmer densities and comparing within and between both haplotypes (smallest SDR identified to-date has been ~5Mb).
The current implementation of this tool uses meryl to count and negate kmers from two genomic haplotypes. Previous implementations relied on multiple piecemeal programs that tooks upwards of 5 hours to complete, the current version (0.2) should take ~30 minutes from takeoff to touchdown.
Running on the a test dataset on a cluster with a 24 core/36Gb RAM allocation reported these times upon successful completion:
time snakemake --use-conda -c 24 -s SCINKD/SCINKD.v2.0.beta.snakefile
real 35m2.348s
user 111m28.608s
sys 14m56.227s
To install:
git clone https://github.com/DrPintoThe2nd/SCINKD.git
mamba create -n scinkd meryl=1.4.1 snakemake=7.32.4 pigz r r-dplyr r-ggplot2 samtools --yes
mamba activate scinkd
File naming restriction: Both input haplotype fasta files MUST be gzipped (or bgzipped) and MUST end in ".hap1.fasta.gz" and ".hap2.fasta.gz" (or their symbolic link does).
WARNING Due to current limitations in the final step of the workflow, runtime scales linearly with the total number of sequences in each haplotype.
Disclaimer This technique reports phasing differences between haplotypes, including contaminants, it's important to look deeper into any regions of interest.
For the test dataset provided (https://doi.org/10.6084/m9.figshare.27040678.v2), this could be applied simply via:
ln -s Anniella_stebbinsi_HiFi_2024.asm.hic.hap1.p_ctg.FINAL.Genbank.fasta.gz Anniella_stebbinsi_HiFi_2024.asm.hic.hap1.fasta.gz
ln -s Anniella_stebbinsi_HiFi_2024.asm.hic.hap2.p_ctg.FINAL.Genbank.fasta.gz Anniella_stebbinsi_HiFi_2024.asm.hic.hap2.fasta.gz
Then, ensure the config.json file reads:
{
"prefix": "Anniella_stebbinsi_HiFi_2024.asm.hic"
}
To run the pipeline on the provided Anniella genome on a machine with 16 available threads (and the default setting of 16Gb of available RAM):
snakemake --use-conda -np -s SCINKD/SCINKD.v2.0.1.beta.snakefile #dry-run to test inputs
snakemake --use-conda -c 16 -s SCINKD/SCINKD.v2.0.1.beta.snakefile #run SCINKD
Chromosome lengths can be calculated using samtools faidx (column two of the fasta index file):
samtools faidx Anniella_stebbinsi_HiFi_2024.asm.hic.hap1.fasta.gz
samtools faidx Anniella_stebbinsi_HiFi_2024.asm.hic.hap2.fasta.gz
Template code used in generating these plots is enclosed (Anniella_template.R) and test files useful for replicating these plots are available alongside the test dataset (https://doi.org/10.6084/m9.figshare.27040678.v2).
Downstream plotting establishes the linear relationship between chromosome length and number of haplotype-specific kmers, as well as the sex chromosomes that significantly deviate from this expectation:
Kmer densities on the Z and W are observably higher:
Regions of increased kmer dentities converge on a single part of the chromosome, syntenic with chicken chromosome 11.
[last two steps in the pipeline need to be replaced with a faster python script]
[additional plotting functions to be added]
[additional documentation to be added]
After implicating a linkage group as a putative sex chromosome, additional anaylses still need to be conducted to validate. I'd recommend starting with a 1:1 haplotype alignment and diving deeper into that using a program like pafr https://github.com/dwinter/pafr or SVbyEye (shown) https://github.com/daewoooo/SVbyEye:
