Introduction

LongTranscriptomics is a nextflow pipeline that is used for the processing of direct RNA nanopore sequencing data, providing multiple transcript reconstruction options, and quantification with the use of a reference genome, and transcriptome annotation.

The pipeline currently only accepts sequencing data from directRNA Oxford Nanopore Technologies (ONT) libraries. It is recommended to provide raw FASTQ files to the pipeline, however, it will also accept already mapped sequencing reads in BAM format. These are provided to the samplesheet as input.

1. QC of FASTQ input files ( NANOQ, SEQUALI )
2. Mapping to reference genome ( minimap2 )
3. Sort and index alignments ( samtools )
4. Create bigWig coverage files ( bedtools, bedGraphToBigWig )
5. Extensive QC of alignments
   1. samtools
   2. cramino
   3. alfred
   4. ngs-bits
6. Multiple transcriptome reconstruction options, with read correction options.
   1. FLAIR - allows read correction
   2. bambu - very minor read correction
   3. IsoQuant - allows read correction
   4. StringTie
7. Transcriptome assessment gffcompare
8. Transcript quantification
   1. oarfish )

Usage

Note

If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

sample,replicate,sequencing_summary_path,read_path
CONTROL1,1,data/long_reads_sequencingsummary_1.txt,data/long_reads_1.fastq.gz
CONTROL1,2,data/long_reads_sequencingsummary_2.txt,data/long_reads_2.fastq.gz

Each row represents a fastq file. Replicate refers to a technical replicate, biological replicates should be named uniquely. Be sure to pay attention to sample naming, in order to avoid duplication and file overwriting.

The basic reference files required to run the pipeline are 1) a genome in fasta format, and 2) a transcriptome annotation in gtf format. It is advised that the files be gzipped, but it is fine if they are not.

Warning

Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

Once you've created the samplesheet, acquired a genome fasta and a transcriptome annotation, you can run the pipeline using:

nextflow run . \
   -profile <docker/singularity/.../institute> \
   -c <CONFIG FILE> \
   --input <SAMPLESHEET> \
   --outdir <OUTDIR> \
   --genome_fasta <GENOME FASTA> \
   --annotation_gtf <ANNOTATION GTF>

To run a minimal, quick test dataset, use:

mkdir testing_dir

nextflow run . \
    -profile test,singularity \
    --outdir testing_dir \
    -c conf/test.config`

For additional documentation on usage of the workflow, and details on outputs, please refer to the usage documentation.

Credits

number-25/LongTranscriptomics was originally written by Dean Bašić.

Citations

If you use number-25/LongTranscriptomics for your analysis, please cite it using the following doi: 10.5281/zenodo.17082314-1073c8.

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.