ONTmetacriptom-NF

A Nextflow Pipeline for Oxford Nanapore Technology(ONT) Long Reads Metatrascriptomic Data Analysis

Introduction

ONTmetacriptom-NF is a bioinformatics analysis pipepliene used for the analysis of Oxford Nanopore Technology long reads metatrascriptomic data. The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructure in a very portable manner. It uses docker and singularity contantainers making installation inconsequential and results highly reproducible.

Pipeline summary

ONTmetacriptom-NF pipeline performs by default the following:

• FastQ reads quality control
• Adaptor removal
• Sorting of ribosomal RNA and Messenger RNA
• Clustering of metatranscriptomes
• Quantification of transcripts and counting reads mapping to host

Components

ONTmetacriptom-NF uses the following software components and tools:

• python=3.9.13
• pip=22.1.2
• NanoPlot 1.40.0
• mutiqc 1.13
• porechop 0.2.4
• samtools 1.6
• sortmerna 4.3.4
• isonclust 0.0.6.1
• spoa 4.0.7
• isoncorrect 0.0.8
• minimap2 2.17
• NanoCount 1.0.0post6

Quick Start

1. Install Nexflow (>=22.04.5)
2. Install any of Docker or Singularity
3. Download the pipeline and test it on a minimal dataset in a single command:

nextflow run main.nf

4. To run the pipeline with your dataset, use the command:

nextflow run main.nf --readsDir '/path/to/dir_Containing_your_barcodes'

To run a pipeline from a GitHub repository,

1. You can run it by specifying the project name as shown below to test the pipeline:

nextflow run eanbit-rt/ONTmetacriptom-NF

2. To run the pipeline with your dataset.

nextflow run eanbit-rt/ONTmetacriptom-NF --readsDir '/path/to/dir_Containing_your_barcodes'

It automatically downloads it and store in the $HOME/.nextflow folder.

Use the command info to show the project information, e.g.:

nextflow info eanbit-rt/ONTmetacriptom-NF

Credits

We thank the following people for their extensive assistance in the development of this pipeline (in alphabetical order):

• Fedrick Kebaso
• Samuel Oduor
• Stephen Kuria
• Manase Aloo
• Mark Tefero Kivumbi

References

These literatures shed more light on the background theories and concepts that were adapted to advance our methodology.

1. Shakya, M., Lo, C. C., & Chain, P. S. G. (2019). Advances and challenges in metatranscriptomic analysis. Frontiers in Genetics, 10(SEP), 904. https://doi.org/10.3389/FGENE.2019.00904/BIBTEX
2. Sahlin, K., Sipos, B., James, P. L., & Medvedev, P. (2021). Error correction enables use of Oxford Nanopore technology for reference-free transcriptome analysis. Nature Communications, 12(1). https://doi.org/10.1038/S41467-020-20340-8
3. Joshua Batson, Gytis Dudas, Eric Haas-Stapleton, Amy L Kistler, Lucy M Li, Phoenix Logan, Kalani Ratnasiri, Hanna Retallack (2021) Single mosquito metatranscriptomics identifies vectors, emerging pathogens and reservoirs in one assay eLife 10:e68353 https://doi.org/10.7554/eLife.68353