# Wf-ToulligQC: A User-Friendly Solution for Quality Control of Nanopore sequencing data on EPI2ME Labs

Ali Hamraoui, Karine Dias, Sophie Lemoine, Morgane Thomas-Chollier, Stéphane Le Crom, Laurent Jourdren

► To cite this version:

Ali Hamraoui, Karine Dias, Sophie Lemoine, Morgane Thomas-Chollier, Stéphane Le Crom, et al. Wf-ToulligQC: A User-Friendly Solution for Quality Control of Nanopore sequencing data on EPI2ME Labs. Assemblée générale France Génomique, Jun 2023, Paris, France. . hal-05142806

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https://hal.science/hal-05142806v1

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# Wf-ToulligQC : A User-Friendly Solution for Quality Control of Nanopore sequencing data on EPI2ME Labs

Ali Hamraoui¹, Karine Dias¹, Sophie Lemoine¹, Morgane Thomas-Chollier¹, Stéphane Le Crom¹,², and Laurent Jourdren¹

1. GenomiqueENS, Institut de biologie de l'ENS (IBENS), Département de biologie, École normale supérieure, CNRS, INSERM, Université PSL, 75005 Paris, France

2. Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Laboratory of Computational and Quantitative Biology (LCQB), 75005 Paris, France

## Introduction

The sequencing devices developed by Oxford Nanopore Technologies (ONT) produce **long DNA sequences** (> 200 kb) and **full-length RNAs**. Sequencing and primary data acquisition are driven by the MinKNOW software, developed by ONT. MinKNOW stores the raw signal data as Fast5 files. Basecalling is then performed either during or after the acquisition step. Basecalling is usually achieved by Guppy or Dorado, the official ONT basecallers. The output sequence reads are stored in FASTQ or Fast5 format.

MinKNOW produces a Quality Control (QC) report as a PDF file at the end of the run. However this report only provides estimated information as it is based on non-basecalled and non-demultiplexed data. Since 2016, GenomiqueENS develops **ToulligQC** [1], a software that performs quality-control for all types of runs from Oxford Nanopore Technologies devices, including multiplexed data. Here, we present the integration of ToulligQC into **EPI2ME Labs** [2], launching and executing ToulligQC through a user-friendly graphical interface has become accessible.

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## Implementation

We introduce **wf-ToulligQC** [3] an open-source workflow (GPL licence) that leverages ToulligQC 2.4, the latest version of our QC software. Implemented in **Nextflow** [4], Wf-ToulligQC offers straightforward install on **EPI2ME Labs**. It simplifies routine Quality Control of ONT runs by enabling users to run ToulligQC within a desktop application with an easy-to-use **graphical interface**.

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EPI2ME

Wf-ToulligQC is available on GitHub, providing a powerful solution for quality control in genomics research. Its nextflow implementation and seamless integration with **container technologies** offer excellent software package management and scalability for **clusters** and **cloud-scale** installations.

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GenomiqueENS/wf-toulligqc

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## Sequencers, flowcells, and run type support

Wf-ToulligQC **supports all** existing Oxford Nanopore **sequencers and flowcells**. It can deal with most of the available protocols such as 1D, directRNA, and soon **Duplex**. The software **requires Guppy** to work. The support of the Albacore basecaller has been removed since ToulligQC 2.0, and the next ONT production basecaller, **Dorado**, will be handled once it reaches general availability. **wf-ToulligQC** will also support input from Dorado, which produces unmapped **BAM** files by default, as well as **FASTQ** files.

<table><caption>Table 1: ToulligQC supported flowcells, protocols and basecallers. Legend: ✓ Supported, ❌ not supported anymore, ■ coming soon.</caption><thead><tr><th></th><th>Flowcells</th><th>Flongle</th><th>MinION</th><th>PromethION</th></tr></thead><tbody><tr><th>Protocols</th><td>1D</td><td>✓</td><td>✓</td><td>Duplex</td></tr><tr><th>Basecallers</th><td>Albacore</td><td>✓</td><td>Guppy</td><td>Dorado</td></tr><tr><th>Input files</th><td>Seq summary</td><td>✓</td><td>FASTQ</td><td>BAM</td></tr></tbody></table>

## References

[1] https://github.com/toulligQC

[2] https://labs.epi2me.io/

[3] https://github.com/wf-toulligQC

[4] https://github.com/nextflow-io/nextflow

[5] https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

## Run overview

Our tool produces a single **HTML report file** inspired by FastQC [5], gathering the graphics and statistics required to estimate run quality. The first part of the report contains three tables about overall statistics, run metadata, devices and software.

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ToulligQC makes it possible to follow the read classification and compile the number of raw, passed, failed and demultiplexed reads. The data can be easily visualised on a read-count histogram, a table or on a plot through time.

Read count histogram ①

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Yield plot through time ①

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**Graphs** generated by ToulligQC are interactive. e.g, the distribution of read lengths can be toggled in log or linear mode, and the PHRED score distribution can be shown on a boxplot or on violin plot.

Distribution of read lengths ①

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PHRED score distribution ①

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The version 2 of ToulligQC can handle data from **all existing Oxford Nanopore flowcells** and can produce a real overview of channel usage over the entire flowcell.

Channel occupancy of the flowcell ①

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Channel occupancy of the flowcell ①

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Channel occupancy of the flowcell ①

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Many graphs about run overview not shown on this poster are available in ToulligQC reports. We regularly enrich ToulligQC with new features to provide the most relevant information for users.

## Barcoded samples

Oxford Nanopore Technology gives the opportunity to sequence multiple samples in one run. Each sample is identified by its barcode (added during library preparation). ToulligQC allows to retrieve each sample proportion in the sequencing run thanks to data provided by Guppy while demultiplexing. It also gives an insight on the read size and quality for each sample independently.

Pass barcoded reads distribution ①

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Pie chart Histogram

Read size distribution for barcodes ①

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