77 lines
3.2 KiB
Markdown
77 lines
3.2 KiB
Markdown
# Pipeline for annotating genomes using long read transcriptomics
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This repository contains a [nextflow](https://www.nextflow.io/) workflow
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for assembly and annotation of transcripts from Oxford Nanopore cDNA or direct RNA reads.
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## Overview
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* cDNA or direct RNA reads are optionally preprocessed by [pychopper](https://github.com/nanoporetech/pychopper) for trimming and orientation. <br>
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* Reads are then mapped to a supplied reference genome using [minimap2](https://github.com/lh3/minimap2) <br>
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* Transcripts are assembled by[stringtie](http://ccb.jhu.edu/software/stringtie) in long read mode (with or without a guide reference annotation) to generate the GFF annotation.
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* The annotation generated by the pipeline is compared to the reference annotation (if supplied) using [gffcompare](http://ccb.jhu.edu/software/stringtie/gffcompare.shtml)
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* An html report is generated, which contains various summary statistics and plots of the data.
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## Quickstart
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The workflow uses [nextflow](https://www.nextflow.io/) to manage compute and
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software resources, as such nextflow will need to be installed before attempting
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to run the workflow.
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The workflow can currently be run using either
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[Docker](https://www.docker.com/products/docker-desktop),
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[Singularity](https://sylabs.io/singularity/) or
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[conda](https://docs.conda.io/en/latest/miniconda.html) to provide isolation of
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the required software. Each method is automated out-of-the-box provided
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either docker, singularity or conda is installed.
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It is not required to clone or download the git repository in order to run the workflow.
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For more information on running EPI2ME Labs workflows [visit out website](https://labs.epi2me.io/wfindex).
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**Workflow options**
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To obtain the workflow, having installed `nextflow`, users can run:
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```
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nextflow run epi2me-labs/wf-isoforms --help
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```
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to see the options for the workflow.
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**Workflow inputs**
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- Directory containing cDNA/direct RNA reads (or path to single file) in fastq/fastq.gz format
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- Reference genome in fast format
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- Optional reference annotation in GFF2/3 format
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**Example workflow run**
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```
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# To run a small and quick example using synthetic data
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nextflow run wf-isoforms/ --fastq test_data/fastq --ref_genome genome.fasta --ref_annotation reference.gff
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--out_dir outdir/ -profile conda
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```
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```
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# To evaluate the workflow on a larger Drosophila dataset
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chmod u+x ./run_evaluation_dmel.sh outdir
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```
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**Workflow outputs**
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* wf-isoforms-report.html
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- Summary and plots of reads, alignments and the transcript assembly and annotation
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* str_merged.gff
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- The stringtie-generated transcript annotations
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* str_merged.stats
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- gffcomare file with statistics regarding the accuracy of the assembled query transcripts in relation to the reference annotation
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* str_merged.annotated.gtf
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- A gffcomapre output file with extra columns relating to comparison with the reference annotation
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* str_transcriptome.fas
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- A transcriptome made from the query reads
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* merged_transcriptome.fas
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-A transcriptome made from the combined query reads and reference annotation
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## Useful links
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* [nextflow](https://www.nextflow.io/)
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* [docker](https://www.docker.com/products/docker-desktop)
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* [Singularity](https://sylabs.io/singularity/)
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* [conda](https://docs.conda.io/en/latest/miniconda.html)
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