This protocol describes how to carry out native telomere sequencing method using Oxford Nanopore Technologies. Telo-Seq delivers single-telomere resolution at high throughput, enabling the multiplexing from 4 to up to 12 samples in a single run. The method captures the telomeric ends (including the sub-telomeric regions) as long fragments, allowing for chromosome arm placement.

It is designed to be used with custom-ordered telomere sequence adapters and custom-ordered telomere splints. The protocol leverages the Native Barcoding Auxiliary Kit V14 (EXP-NBA114), the Sequencing Auxiliary Vials V14 (EXP-AUX003), and the Ultra-Long Auxiliary Vials (EXP-ULA001).

For more information on telomere sequencing and the method described in this protocol, please see our Telo-Seq know-how document.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Extract your DNA, and check its length, quantity and purity. Performing quality checks on your DNA prior to its use in Telo-Seq is essential in ensuring experimental success.
• Ensure you have your sequencing kit, the correct equipment and third-party reagents.
• Ensure you have ordered the required custom-ordered oligos for telomere adapters and splints.
• Download the software for acquiring and analysing your data.
• Check your flow cell to ensure it has enough pores for a good sequencing run.

Library preparation

The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.

Sequencing and analysis

You will need to:

• Start a sequencing run using the MinKNOW software which will collect raw data from the device.
• Basecall and demultiplex your sequencing data using Dorado and command line.
• Analyse your Telo-Seq data using the wf-teloseq workflow via EPI2ME or command line to count telomere reads and estimate telomere length.

For example:

Note: Please note that an input of less than 60 µg into the library preparation will not yield sufficient DNA library to fill your flow cell, resulting in reduced telomeric output.

Additionally, we recommend performing at least a 4-plex even if the DNA of from the same origin. If you are only running one sample, or subsets smaller than 4 samples, we recommend splitting these across at least 4 barcodes with 15 µg input into each.

Please refer to the input mass and multiplexing section of the Telo-Seq know-how document for more details.

Input DNA fragment distribution

Optimal performance is observed in samples with longer mean fragment size.

We have found that the presence of fragments <10 kb is detrimental to performance, while samples with a mean fragment size >20 kb will perform well.

If there is a high proportion (>10%) of fragments <10 kb in your sample(s), we recommend performing a  size selection of HMW DNA using Short Fragment Eliminator (EXP-SFE001) method. Please refer to the correcting sub-optimal fragment distribution section of the Telo-Seq know-how document for more details.

We recommend using the following methods for DNA extraction for use in with the telo-seq protocol:

• Qiagen® Puregene Cell Kit (Qiagen, 158043)
• Monarch® HMW DNA Extraction Kit for Cells & Blood (NEB, T3050) using an agitation speed of 300 rpm to preserve longer DNA fragments.

We do not recommend the following extraction methods for use with the Telo-seq protocol, as they yield DNA with a high proportion of fragments smaller than 10 kb, leading to reduced performance:

• Qiagen DNeasy Blood & Tissue Kit (Qiagen, 69504)
• Qiagen Genomic-tip (Qiagen, 10243)

Please note, alternative methods are available but have not been tested by Oxford Nanopore Technologies.

Please refer to the achieving optimal fragment distribution section of the Telo-Seq know-how document for more details.

How to QC your input DNA It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Chemical contaminants Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

Additional Agencourt AMPure XP beads are required alongside the AMPure XP Beads (AXP) provided in the Native Barcoding Auxiliary Kit V14 (EXP-NBA114) for the digestion and dA-tailing of non-telomeric gDNA and the splint annealing and clean-up steps of the protocol.

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

Order the following HPLC-purified oligos. Resuspend to 100 μM in TE buffer for use in the protocol:

Note: The barcoded telo-adapters and telo-splints are only compatible with their corresponding numbered counterpart (e.g. TA01 with TS01, TA02 with TS02, etc). Please ensure you are using the correct barcoded pairs during the pre-hybridisation steps.

Telo-adapters (TA01 - TA12)

Telo-splints (TS01 - TS12)

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION. Oxford Nanopore Technologies will replace any unused flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

This protocol uses the Native Barcoding Auxiliary Kit V14 (EXP-NBA114), Long Fragment Buffer Expansion (EXP-LFB001), Ultra-Long Auxiliary Vials (EXP-ULA001), and Sequencing Auxiliary Vials V14 (EXP-AUX003) for library preparation and flow cell priming.

Native Barcoding Auxiliary Kit V14 (EXP-NBA114)

Note: This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: For the Telo-Seq method you will not require the Short Fragment Buffer (SFB) from the Native Barcoding Auxiliary Kit V14 (EXP-NBA114). If you do not use this component for other methods it can be disposed of.

LFB Expansion (EXP-LFB001)

Ultra-Long Auxiliary Vials (EXP-ULA001)

Note: For the Telo-Seq method you will not require the Sequencing Buffer UL (SBU) or the Loading Solution UL (LSU) from the Ultra-Long Auxiliary Vials (EXP-ULA001). If you do not use these components for other methods they can be disposed of.

Sequencing Auxiliary Vials V14 (EXP-AUX003)

Note: For the Telo-Seq method you will not require the Library Solution (LIS) or the Flow Cell Tether (FCT) from the Sequencing Auxiliary Vials V14 (EXP-AUX003). If you do not use these components for other methods they can be disposed of.

For example:

Note: Please note that an input of less than 60 µg into the library preparation will not yield sufficient DNA library to fill your flow cell, resulting in reduced telomeric output.

Additionally, we recommend performing at least a 4-plex even if the DNA of from the same origin. If you are only running one sample, or subsets smaller than 4 samples, we recommend splitting these across at least 4 barcodes with 15 µg input into each.

Please refer to the input mass and multiplexing section of the Telo-Seq know-how document for more details.

The sequencing device control and data acquisition are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.

We recommend setting up your sequencing run using the recommendations outlined below. All other parameters can be left to their default settings.

Basecalling of sequencing data will be performed post-sequencing using the wf-teloseq pipeline.

To produce MinKNOW sequencing metrics during your sequencing run, you should use the Fast basecalling model. Please note, this is only used for the sequencing metrics and all data generated will need to be re-basecalled after sequencing.

Below are the recommendations for MinKNOW settings:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-PRO114M

Sample ID: [user defined]

Kit

Kit selection: Ligation Sequencing Kit (SQK-LSK114)

Run configuration

Sequencing and analysis

Basecalling: On
Modified bases: Off [default]
Model: Fast basecalling (for sequencing metrics)

Barcoding: Off [default]

Alignment: Off [default]

Adaptive sampling: Off [default]

Advanced options
Time between pore scans: 1.5 [default]
Reserve pores: On [default]

Data targets

Run limit: Stop run when sequencing reaches 48 hours

Output

Output format
.BAM: Off
.FASTQ: Off
Raw reads: On [default]
.POD5: On [default]

Filtering: On [default]
Qscore: 8 [default]
Minimum read length: 200 b

Telo-Seq data can be analysed using wf-teloseq, a bioinformatics workflow implemented in Nextflow that is part of Oxford Nanopore’s EPI2ME suite of analysis workflows. wf-teloseq can be run from the command line or within the EPI2ME Desktop App. Prior to analysis, the data must be basecalled and demultiplexed using a script available as part of wf-teloseq.

Note: To ensure the compute on your device can keep up with the requirements for sequencing and/or analysis, we strongly recommend against running both processes at the same time.

The script outlined below runs Oxford Nanopore’s Dorado basecaller and demultiplexes the basecalled reads using the barcoded Telo-adapter sequences. The script requires Dorado to be installed and the location available in the command line path. For first time installation of Dorado please refer to the installing Dorado guidelines in the troubleshooting section of this protocol.

To basecall and correctly demultiplex your Telo-Seq data complete the following steps:

1. Download the wf-teloseq pipeline from the GitHub repository

2. Execute the following command in command line:

/path/to/wf-teloseq/bin/basecall.sh -m sup -i /path/to/pod5_files -o /path/to/output

• -m is the basecalling model to use (‘hac’ or ‘sup’)
• -i is path to folder with sequencing data (.pod5)
• -o is the folder where basecalling and demultiplexing results will be output.
• Relative or absolute paths to files can be used.

After successful execution, the output directory will contain a directory for each barcode (barcode01-barcode12, corresponding to the 12 Telo-adapter sequences) containing demultiplexed, basecalled reads (.bam). Any reads not assigned to a barcode will be output to the unclassified folder.

If not already installed on your device, you can download and install the EPI2ME Desktop App following these instructions. If you are running the wf-teloseq for the first time, you can import the workflow by selecting wf-teloseq under the Available Workflows tab within EPI2ME Desktop.

To run the workflow:

1. Launch the EPI2ME Desktop App.

2. Select wf-teloseq under the Installed workflows tab. Then select Run this workflow and Run on your computer.

3. Under Input options, provide individual bam file location in the BAM box. You may provide a single sample file, or a set of folders together with a sample sheet (Sample Options). You may also use the sample sheet to provide sample-specific references if not the same for all barcodes.

4. Optional: Under Teloseq Options, check Skip mapping for a reference-free approach to report global telomere length estimation only.

5. Optional: To adjust default values for minimum read length (default 100) and minimum read quality (default 9), change values under Advanced Options section.

6. Click Launch workflow.

You may adjust values under Teloseq Options and Nextflow configuration to optimise the workflow run according to the compute available to you.

Example wf-teloseq run configuration in the EPI2ME Desktop App:

To execute wf-teloseq from the command line, run the following command:

nextflow run epi2me-labs/wf-teloseq \ 
--bam 'path/to/data' \
--reference 'path/to/reference.fasta.gz' \
-profile standard 

Where --bam is a path to either a bam file or a directory of bam files and --reference is a path to the reference genome of the sequenced sample. If no reference is provided, the workflow will default to a human telomere reference extracted from the HG002 telomere-to-telomere assembly by the T2T Consortium (v1.0.1 release, available from GitHub). For a reference-free approach that analyses whole sample telomere length only, use --skip_mapping and omit the --reference parameter .

Nextflow and either Docker or Singularity must be installed on your system to run the workflow. See the EPI2ME Quick Start guide for additional information on how to install and run EPI2ME workflows from the command line. See the wf-teloseq GitHub page for more detailed information on input parameter options, including how to specify barcode specific reference files if multiplexing more than one sample type.

wf-teloseq produces an interactive HTML report with information on data quality, filtering metrics, and tables and graphs on estimated telomere lengths (Figure 3). The workflow also outputs additional files that can be used for downstream analysis and exploration, including alignment files (.bam) for inspecting the data with a genome browser and chromosome arm resolution telomere read metrics (.tsv).

wf-teloseq output report. (a) Basic data quality metrics (b) Global telomere length estimation (c) Estimated telomere lengths per chromosome arm:

For optimal performance we recommend using a PromethION device and flow cells to sequence your Telo-Seq libraries as this will maximise the output. However, sequencing can also be performed on MinION and GridION devices using minION flow cells.

The table below illustrates example yields from identical libraries sequenced on the two platforms:

Telo-Seq development and validation at Oxford Nanopore Technologies have primarily used high HMW genomic DNA extracted from GM24385 cell cultures. The below table shows typical expected performance of a 12-plex Telo-Seq run using 5 µg input DNA per barcode on mixed human and cow samples. This dataset is publicly available on Oxford Nanopore Technologies open data portal.

Representative outputs of a 12-plex Telo-Seq performed using HMW gDNA extracted from human and cow samples and sequended for 48 hours:

A representative read length distribution on a MinKNOW report for a Telo-Seq run on a PromethION:

A representative Q score distribution over 48 hours of sequencing on a MinKNOW report for a Telo-Seq run on a PromethION:

Representative pore activity over 48 hours of sequencing on a MinKNOW report for a Telo-Seq run on a PromethION. It is expected that a proportion of pores will remain ‘Open’ (dark green) and available for sequencing for the duration of the run:

A representative pore scan graph on a MinKNOW report for a Telo-Seq run on a PromethION. It is not unusual for the health of the flow cell to deteriorate more rapidly than with non-Telo-Seq experiments:

Representative translocation speeds and temperature over 48 hours of sequencing on a MinKNOW report for a Telo-Seq run on a PromethION:

Re-using these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.