Introduction

nf-core/txmupdate is a bioinformatics pipeline that can be used refine reference transcriptome annotations utilizing RNA sequencing data. It takes a samplesheet and FASTQ files as input, performs quality control (QC), trimming and STAR alignment, BAM processing and normalization, and a GFF comparison/insertion step.

0. Standardize GTF/GFF input format AGAT
1. Merge re-sequenced FastQ files (cat)
2. Sub-sample FastQ files and auto-infer strandedness (fq, Salmon)
3. Read QC (FastQC)
4. UMI extraction (UMI-tools)
5. Adapter and quality trimming (Trim Galore!)
6. Removal of ribosomal RNA (SortMeRNA)
7. Read Alignment to Reference STAR
8. Normalize coverage depth of alignemnts Bamsifter
9. Sort, merge, and index alignments (SAMtools)
10. Generate trancsript products from alignments (StringTie)
11. Compare transcript constructs against reference (GFFCompare
12. Transcript insertion - GTF/GFF re-writing GTF_INSERT
13. Pseudo-alignment and quantification (Salmon; optional)

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,fastq_1,fastq_2,strandedness
CONTROL_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz,auto
CONTROL_REP1,AEG588A1_S1_L003_R1_001.fastq.gz,AEG588A1_S1_L003_R2_001.fastq.gz,auto
CONTROL_REP1,AEG588A1_S1_L004_R1_001.fastq.gz,AEG588A1_S1_L004_R2_001.fastq.gz,auto

Each row represents a fastq file (single-end) or a pair of fastq files (paired end). Rows with the same sample identifier are considered technical replicates and merged automatically. The strandedness refers to the library preparation and will be automatically inferred if set to auto.

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.

Now, you can run the pipeline using:

nextflow run nf-core/txmupdate \
    --input samplesheet.csv \
    --outdir <OUTDIR> \
    -profile <docker/singularity/.../institute>

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

You can cite the nf-core publication as follows:

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.