B-Chromosome Detection Pipeline Using Reference-Derived k-mers

This repository contains a computational pipeline for identifying and estimating the copy number of B chromosomes (chrB) in Distichlis whole-genome sequencing (WGS) datasets using chromosome-specific k-mers.

The workflow builds a high-confidence database of B chromosome-specific k-mers from reference genomes and screens thousands of WGS samples to predict B chromosome copy number. Predictions can be validated experimentally using fluorescence in situ hybridization (FISH).

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Workflow Overview

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Pipeline

Step 1. Assemble B chromosomes (chrB)

High-quality Distichlis genome assemblies are used to identify and curate B chromosome sequences.

Quality control includes:

• Removing Ns and assembly gaps
• Retaining the longest chrB contigs
• Standardizing chromosome orientation
• Annotating repeat regions and telomeres

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Step 2. Build candidate chrB k-mers

The curated B chromosome sequences are converted into 51-mers using Jellyfish.

Candidate chrB k-mers are obtained by intersecting the k-mer sets from multiple independently assembled B chromosomes.

Output:

• Common chrB k-mers shared among reference genomes

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Step 3. Remove A chromosome k-mers

To ensure high specificity, candidate chrB k-mers are compared against the A chromosomes from all reference genomes.

Any k-mer found in an A chromosome is removed.

Output:

• High-confidence B chromosome-specific k-mer database (DBk)

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Step 4. Screen WGS samples

Each WGS sample is queried against the chrB-specific k-mer database.

For sample i,

[ K_i=\frac{C_i}{N_i-k+1}\times10^9 ]

where

• (C_i) = number of chrB-specific k-mer hits
• (N_i) = total sequenced bases
• (k=51)

The normalized value is converted into an estimated B chromosome copy number using reference distributions from B-negative samples.

Example classification:

Predicted value	Classification
<0.5	0B
0.5–1.5	1B
1.5–2.5	2B
...	...

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Step 5. Experimental validation

Predicted copy numbers are validated using fluorescence in situ hybridization (FISH).

Two probe sets are designed:

• chrB-specific probes
• Centromere-specific probes

FISH is performed on root tip or meiotic cells and compared with computational predictions.

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Input

• High-quality Distichlis reference genomes
• Curated B chromosome assemblies
• Whole-genome sequencing reads (FASTQ)

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Output

• chrB-specific k-mer database
• Normalized chrB k-mer counts
• Predicted B chromosome copy number
• FISH validation results

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Software

• Jellyfish
• Python
• Bash
• BEDTools (optional)
• SAMtools (optional)

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Applications

• Large-scale B chromosome screening
• Population genetics
• Cytogenomics
• Evolutionary genomics
• High-throughput B chromosome detection

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Citation

If you use this pipeline, please cite the associated publication once available.