TeloTracker

A pipeline for long-read sequencing data of yeast telomeres.

TeloTracker is a Python package designed to track and visualize long read sequencing data (Oxford Nanopore Technology (ONT) and PacBio) sequencing of yeast telomeres. TeloTracker was specifically developed to track and analyze the development of Alternative Lengthening of Telomeres in yeast, but the program can be appied/adapted to follow the dynamics of yeast telomeres in general.

Installation

To install TeloTracker, you can clone this repository by running the command below.

git clone https://github.com/Jacob-M-Wells/TeloTracker.git
cd TeloTracker

Installing with conda (Recommended)

conda env create -n telotracker -f environment.yml
conda activate telotracker

Installing Dorado (Required if doing basecalling)

Dorado is a high-performance, open source basecaller for Oxford Nanopore reads. Dorado can be run with a docker container or installed locally.

Dorado must be used to basecall (rather than using MinKnow), as basecalling without trimming adapters/barcode sequences is required.

📝 Detailed instructions and usage examples are available in the Dorado documentation.

Option 1. Run with Docker Container (using singularity)

📦 Download the latest release for your platform from the Dorado Docker Releases page.

singularity pull dorado.sif docker://nanoporetech/dorado:latest
singularity exec dorado.sif dorado --help

Option 2. Run with local installation

📦 Download the latest release for your platform from the Dorado GitHub Releases page. The current version as of 03/13/2025: Dorado 0.9.1 @ https://cdn.oxfordnanoportal.com/software/analysis/dorado-0.9.1-linux-x64.tar.gz

wget https://cdn.oxfordnanoportal.com/software/analysis/dorado-0.9.1-linux-x64.tar.gz
gunzip https://cdn.oxfordnanoportal.com/software/analysis/dorado-0.9.1-linux-x64.tar.gz
tar -xvf https://cdn.oxfordnanoportal.com/software/analysis/dorado-0.9.1-linux-x64.tar.gz
mv dorado-0.9.1-linux-x64/ ~/bin/
ln -s ~/bin/dorado-0.9.1-linux-x64/bin/dorado ~/bin/dorado
source ~/.bashrc
dorado --help

Overview

TeloTracker processes long-read sequencing data (tested with ONT and PacBio) to analyze reads for telomere and sub-telomere features. Run telotracker with telomere_analysis.sh

Running telomere_analysis.sh begins with a core analysis step that identifies basic telomere features (telomere length and Y' element count). If additional reference files are provided/available, then analysis for recombination will be performed.

• Core Analysis : Gets chromosome-end-specific telomere lengths and Y' counts in idividual reads
• Recombination Analysis : Labels Y' elements, X elements, and their recombination points. Requires additional reference files.

Requirements

TeloTracker is designed to run on an HPC cluster (tested on the University of Iowa Argon HPC) but can also be run on a local machine. Adjust thread and memory settings at the top of each script to match your environment.

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Quick Start

1. Activate the environment

conda activate telotracker

2. Run the pipeline

bash telomere_analysis.sh <input> <base_name> <output_dir> <strain_number> [strain_ref_dir]

Examples:

# Core analysis only (steps 0–6)
bash telomere_analysis.sh data/6991_day0_with_selection-subset.fastq.gz 6991_day0 ./results 6991

# Full analysis including recombination (steps 0–10)
bash telomere_analysis.sh data/6991_day0_with_selection-subset.fastq.gz 6991_day0 ./results 6991 references/6991_features

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Arguments

Argument	Required	Description
input	Yes	Path to input .bam, .fastq, or .fastq.gz file
base_name	Yes	Sample name used for all output file naming (e.g. 6991_day0)
output_dir	Yes	Directory where all outputs will be written
strain_number	Yes	Strain identifier (e.g. 6991, 7172, 7302)
strain_ref_dir	No	Path to strain-specific reference directory for steps 7–9 (e.g. references/6991_features). Omit or pass none to run core analysis only.

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Configuration

Open telomere_analysis.sh and adjust the following variables near the top of the file before running:

Variable	Default	Description
THREADS	80	Number of CPU threads for BLAST and RepeatMasker
ANCHOR_SET	telomerase_shutoff_anchors	Anchor set to use. Options: telomerase_shutoff_anchors (for strains 6991 and subsequent transformants) or telomerase_deletion_anchors (for strain 6212)

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Pipeline Steps

Core analysis (steps 0–6)

Step	Description
0	Prepare input — convert BAM/FASTQ, filter reads (≥2000 bp, Q≥10), convert to FASTA
1	BLAST reads against chromosome anchor sequences
2	Filter BLAST results for reads with confirmed chromosome anchors
3	Split and label chromosome-anchored reads by arm
4	Trim adapters with Porechop-ABI; check adapter calls; perform fine telomere trimming
5	BLAST chromosome-anchored reads against Y′ probe sequences (per chromosome arm)
6	Y′ analysis and telomere length plots

Recombination analysis (steps 7–10, requires strain_ref_dir)

Step	Description
7	RepeatMasker — identify Y′ elements in anchored reads; compute recombination stats and Y′ pairings
8	RepeatMasker — identify X element ends in paired reads
9	RepeatMasker — identify spacer sequences in paired reads
10	Determine recombination switch locations

Steps 7–10 are skipped automatically if strain_ref_dir is not provided or the directory does not exist.

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Repository Structure

telotracker/
├── telomere_analysis.sh
├── scripts/                    # Python analysis scripts
└── references/
    ├── universal/              # Shared reference files
    │   ├── nanopore_sqk-slk114_adapter_sequence_truncated.txt
    │   └── y_prime_probe.fasta
    ├── anchors/                # Anchor sequence databases
    │   ├── telomerase_shutoff_anchors.fasta
    │   └── telomerase_deletion_anchors.fasta
    ├── 6991_features/          # Strain-specific reference files
    │   ├── 6991_final_features.bed
    │   ├── repeatmasker_6991_all_y_primes.fasta
    │   ├── 6991_x_element_ends_pairings/
    │   └── 6991_spacer_pairings/
    ├── 7172_features/
    │   ├── 7172_final_features.bed
    │   ├── repeatmasker_7172_all_y_primes.fasta
    │   ├── 7172_x_element_ends_pairings/
    │   └── 7172_spacer_pairings/
    └── 7302_features/
        ├── 7302_final_features.bed
        ├── repeatmasker_7302_all_y_primes.fasta
        ├── 7302_x_element_ends_pairings/
        └── 7302_spacer_pairings/