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txome_refinement

transcriptome refinement leveraging the RNAseq nf-core pipeline

https://github.com/riley-grindle/txome_refinement

Science Score: 44.0%

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Repository

transcriptome refinement leveraging the RNAseq nf-core pipeline

Basic Info
  • Host: GitHub
  • Owner: Riley-Grindle
  • License: mit
  • Language: HTML
  • Default Branch: main
  • Size: 36.5 MB
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  • Stars: 1
  • Watchers: 1
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  • Open Issues: 6
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Created about 2 years ago · Last pushed about 1 year ago
Metadata Files
Readme Changelog Contributing License Code of conduct Citation

README.md

nf-core/rnaseq nf-core/rnaseq

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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.

nf-core/txmupdate metro map

  1. Standardize GTF/GFF input format AGAT
  2. Merge re-sequenced FastQ files (cat)
  3. Sub-sample FastQ files and auto-infer strandedness (fq, Salmon)
  4. Read QC (FastQC)
  5. UMI extraction (UMI-tools)
  6. Adapter and quality trimming (Trim Galore!)
  7. Removal of ribosomal RNA (SortMeRNA)
  8. Read Alignment to Reference STAR
  9. Normalize coverage depth of alignemnts Bamsifter
  10. Sort, merge, and index alignments (SAMtools)
  11. Generate trancsript products from alignments (StringTie)
  12. Compare transcript constructs against reference (GFFCompare
  13. Transcript insertion - GTF/GFF re-writing GTF_INSERT
  14. 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:

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:

bash 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.

Owner

  • Login: Riley-Grindle
  • Kind: user

Citation (CITATIONS.md) 

# nf-core/rnaseq: Citations

## [nf-core](https://pubmed.ncbi.nlm.nih.gov/32055031/)

> Ewels PA, Peltzer A, Fillinger S, Patel H, Alneberg J, Wilm A, Garcia MU, Di Tommaso P, Nahnsen S. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol. 2020 Mar;38(3):276-278. doi: 10.1038/s41587-020-0439-x. PubMed PMID: 32055031.

## [Nextflow](https://pubmed.ncbi.nlm.nih.gov/28398311/)

> Di Tommaso P, Chatzou M, Floden EW, Barja PP, Palumbo E, Notredame C. Nextflow enables reproducible computational workflows. Nat Biotechnol. 2017 Apr 11;35(4):316-319. doi: 10.1038/nbt.3820. PubMed PMID: 28398311.

## Pipeline tools

- [BBMap](https://sourceforge.net/projects/bbmap/)

- [BEDTools](https://pubmed.ncbi.nlm.nih.gov/20110278/)

  > Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010 Mar 15;26(6):841-2. doi: 10.1093/bioinformatics/btq033. Epub 2010 Jan 28. PubMed PMID: 20110278; PubMed Central PMCID: PMC2832824.

- [fastp](https://www.ncbi.nlm.nih.gov/pubmed/30423086/)

  > Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1;34(17):i884-i890. doi: 10.1093/bioinformatics/bty560. PubMed PMID: 30423086; PubMed Central PMCID: PMC6129281.

- [FastQC](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)

- [featureCounts](https://pubmed.ncbi.nlm.nih.gov/24227677/)

  > Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014 Apr 1;30(7):923-30. doi: 10.1093/bioinformatics/btt656. Epub 2013 Nov 13. PubMed PMID: 24227677.

- [fq](https://github.com/stjude-rust-labs/fq)

- [GffRead](https://pubmed.ncbi.nlm.nih.gov/32489650/)

  > Pertea G, Pertea M. GFF Utilities: GffRead and GffCompare. F1000Res. 2020 Apr 28;9:ISCB Comm J-304. doi: 10.12688/f1000research.23297.2. eCollection 2020. PubMed PMID: 32489650; PubMed Central PMCID: PMC7222033.

- [HISAT2](https://pubmed.ncbi.nlm.nih.gov/31375807/)

  > Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019 Aug;37(8):907-915. doi: 10.1038/s41587-019-0201-4. Epub 2019 Aug 2. PubMed PMID: 31375807.

- [MultiQC](https://pubmed.ncbi.nlm.nih.gov/27312411/)

  > Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924.

- [picard-tools](http://broadinstitute.github.io/picard)

- [preseq](https://pubmed.ncbi.nlm.nih.gov/23435259/)

  > Daley T, Smith AD. Predicting the molecular complexity of sequencing libraries. Nat Methods. 2013 Apr;10(4):325-7. doi: 10.1038/nmeth.2375. Epub 2013 Feb 24. PubMed PMID: 23435259; PubMed Central PMCID: PMC3612374.

- [Qualimap 2](https://pubmed.ncbi.nlm.nih.gov/26428292/)

  > Okonechnikov K, Conesa A, García-Alcalde F. Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data Bioinformatics. 2016 Jan 15;32(2):292-4. doi: 10.1093/bioinformatics/btv566. Epub 2015 Oct 1. PubMed PMID: 26428292; PubMed Central PMCID: PMC4708105.

- [RSEM](https://pubmed.ncbi.nlm.nih.gov/21816040/)

  > Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome BMC Bioinformatics. 2011 Aug 4;12:323. doi: 10.1186/1471-2105-12-323. PubMed PMID: 21816040; PubMed Central PMCID: PMC3163565.

- [RSeQC](https://pubmed.ncbi.nlm.nih.gov/22743226/)

  > Wang L, Wang S, Li W. RSeQC: quality control of RNA-seq experiments Bioinformatics. 2012 Aug 15;28(16):2184-5. doi: 10.1093/bioinformatics/bts356. Epub 2012 Jun 27. PubMed PMID: 22743226.

- [Salmon](https://pubmed.ncbi.nlm.nih.gov/28263959/)

  > Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression Nat Methods. 2017 Apr;14(4):417-419. doi: 10.1038/nmeth.4197. Epub 2017 Mar 6. PubMed PMID: 28263959; PubMed Central PMCID: PMC5600148.

- [SAMtools](https://pubmed.ncbi.nlm.nih.gov/19505943/)

  > Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15;25(16):2078-9. doi: 10.1093/bioinformatics/btp352. Epub 2009 Jun 8. PubMed PMID: 19505943; PubMed Central PMCID: PMC2723002.

- [SortMeRNA](https://pubmed.ncbi.nlm.nih.gov/23071270/)

  > Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data Bioinformatics. 2012 Dec 15;28(24):3211-7. doi: 10.1093/bioinformatics/bts611. Epub 2012 Oct 15. PubMed PMID: 23071270.

- [STAR](https://pubmed.ncbi.nlm.nih.gov/23104886/)

  > Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner Bioinformatics. 2013 Jan 1;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25. PubMed PMID: 23104886; PubMed Central PMCID: PMC3530905.

- [StringTie2](https://pubmed.ncbi.nlm.nih.gov/31842956/)

  > Kovaka S, Zimin AV, Pertea GM, Razaghi R, Salzberg SL, Pertea M. Transcriptome assembly from long-read RNA-seq alignments with StringTie2 Genome Biol. 2019 Dec 16;20(1):278. doi: 10.1186/s13059-019-1910-1. PubMed PMID: 31842956; PubMed Central PMCID: PMC6912988.

- [Trim Galore!](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)

- [UMI-tools](https://pubmed.ncbi.nlm.nih.gov/28100584/)

  > Smith T, Heger A, Sudbery I. UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy Genome Res. 2017 Mar;27(3):491-499. doi: 10.1101/gr.209601.116. Epub 2017 Jan 18. PubMed PMID: 28100584; PubMed Central PMCID: PMC5340976.

- [UCSC tools](https://pubmed.ncbi.nlm.nih.gov/20639541/)
  > Kent WJ, Zweig AS, Barber G, Hinrichs AS, Karolchik D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 2010 Sep 1;26(17):2204-7. doi: 10.1093/bioinformatics/btq351. Epub 2010 Jul 17. PubMed PMID: 20639541; PubMed Central PMCID: PMC2922891.

## R packages

- [R](https://www.R-project.org/)

  > R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

- [DESeq2](https://pubmed.ncbi.nlm.nih.gov/25516281/)

  > Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. PubMed PMID: 25516281; PubMed Central PMCID: PMC4302049.

- [dupRadar](https://pubmed.ncbi.nlm.nih.gov/27769170/)

  > Sayols S, Scherzinger D, Klein H. dupRadar: a Bioconductor package for the assessment of PCR artifacts in RNA-Seq data BMC Bioinformatics. 2016 Oct 21;17(1):428. doi: 10.1186/s12859-016-1276-2. PubMed PMID: 27769170; PubMed Central PMCID: PMC5073875.

- [ggplot2](https://cran.r-project.org/web/packages/ggplot2/index.html)

  > H. Wickham. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.

- [optparse](https://CRAN.R-project.org/package=optparse)

  > Trevor L Davis (2018). optparse: Command Line Option Parser.

- [pheatmap](https://CRAN.R-project.org/package=pheatmap)

  > Raivo Kolde (2018). pheatmap: Pretty Heatmaps.

- [RColorBrewer](https://CRAN.R-project.org/package=RColorBrewer)

  > Erich Neuwirth (2014). RColorBrewer: ColorBrewer Palettes.

- [SummarizedExperiment](https://bioconductor.org/packages/release/bioc/html/SummarizedExperiment.html)

  > Morgan M, Obenchain V, Hester J and Pagès H (2020). SummarizedExperiment: SummarizedExperiment container.

- [Tximeta](https://pubmed.ncbi.nlm.nih.gov/32097405/)
  > Love MI, Soneson C, Hickey PF, Johnson LK, Pierce NT, Shepherd L, Morgan M, Patro R. Tximeta: Reference sequence checksums for provenance identification in RNA-seq PLoS Comput Biol. 2020 Feb 25;16(2):e1007664. doi: 10.1371/journal.pcbi.1007664. eCollection 2020 Feb. PubMed PMID: 32097405; PubMed Central PMCID: PMC7059966.

## Software packaging/containerisation tools

- [Anaconda](https://anaconda.com)

  > Anaconda Software Distribution. Computer software. Vers. 2-2.4.0. Anaconda, Nov. 2016. Web.

- [Bioconda](https://pubmed.ncbi.nlm.nih.gov/29967506/)

  > Grüning B, Dale R, Sjödin A, Chapman BA, Rowe J, Tomkins-Tinch CH, Valieris R, Köster J; Bioconda Team. Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018 Jul;15(7):475-476. doi: 10.1038/s41592-018-0046-7. PubMed PMID: 29967506.

- [BioContainers](https://pubmed.ncbi.nlm.nih.gov/28379341/)

  > da Veiga Leprevost F, Grüning B, Aflitos SA, Röst HL, Uszkoreit J, Barsnes H, Vaudel M, Moreno P, Gatto L, Weber J, Bai M, Jimenez RC, Sachsenberg T, Pfeuffer J, Alvarez RV, Griss J, Nesvizhskii AI, Perez-Riverol Y. BioContainers: an open-source and community-driven framework for software standardization. Bioinformatics. 2017 Aug 15;33(16):2580-2582. doi: 10.1093/bioinformatics/btx192. PubMed PMID: 28379341; PubMed Central PMCID: PMC5870671.

- [Docker](https://dl.acm.org/doi/10.5555/2600239.2600241)

- [Singularity](https://pubmed.ncbi.nlm.nih.gov/28494014/)
  > Kurtzer GM, Sochat V, Bauer MW. Singularity: Scientific containers for mobility of compute. PLoS One. 2017 May 11;12(5):e0177459. doi: 10.1371/journal.pone.0177459. eCollection 2017. PubMed PMID: 28494014; PubMed Central PMCID: PMC5426675.

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Dependencies

modules/local/convertspgxf2gxf/meta.yml cpan
modules/nf-core/bbmap/bbsplit/meta.yml cpan
modules/nf-core/cat/fastq/meta.yml cpan
modules/nf-core/custom/dumpsoftwareversions/meta.yml cpan
modules/nf-core/custom/getchromsizes/meta.yml cpan
modules/nf-core/fastp/meta.yml cpan
modules/nf-core/fastqc/meta.yml cpan
modules/nf-core/fq/subsample/meta.yml cpan
modules/nf-core/gffread/meta.yml cpan
modules/nf-core/gunzip/meta.yml cpan
modules/nf-core/hisat2/align/meta.yml cpan
modules/nf-core/hisat2/build/meta.yml cpan
modules/nf-core/hisat2/extractsplicesites/meta.yml cpan
modules/nf-core/picard/markduplicates/meta.yml cpan
modules/nf-core/preseq/lcextrap/meta.yml cpan
modules/nf-core/qualimap/rnaseq/meta.yml cpan
modules/nf-core/rsem/calculateexpression/meta.yml cpan
modules/nf-core/rsem/preparereference/meta.yml cpan
modules/nf-core/rseqc/bamstat/meta.yml cpan
modules/nf-core/rseqc/inferexperiment/meta.yml cpan
modules/nf-core/rseqc/innerdistance/meta.yml cpan
modules/nf-core/rseqc/junctionannotation/meta.yml cpan
modules/nf-core/rseqc/junctionsaturation/meta.yml cpan
modules/nf-core/rseqc/readdistribution/meta.yml cpan
modules/nf-core/rseqc/readduplication/meta.yml cpan
modules/nf-core/rseqc/tin/meta.yml cpan
modules/nf-core/salmon/index/meta.yml cpan
modules/nf-core/salmon/quant/meta.yml cpan
modules/nf-core/samtools/flagstat/meta.yml cpan
modules/nf-core/samtools/idxstats/meta.yml cpan
modules/nf-core/samtools/index/meta.yml cpan
modules/nf-core/samtools/sort/meta.yml cpan
modules/nf-core/samtools/stats/meta.yml cpan
modules/nf-core/sortmerna/meta.yml cpan
modules/nf-core/star/align/meta.yml cpan
modules/nf-core/star/genomegenerate/meta.yml cpan
modules/nf-core/stringtie/stringtie/meta.yml cpan
modules/nf-core/subread/featurecounts/meta.yml cpan
modules/nf-core/trimgalore/meta.yml cpan
modules/nf-core/ucsc/bedclip/meta.yml cpan
modules/nf-core/ucsc/bedgraphtobigwig/meta.yml cpan
modules/nf-core/umitools/dedup/meta.yml cpan
modules/nf-core/umitools/extract/meta.yml cpan
modules/nf-core/untar/meta.yml cpan
subworkflows/nf-core/bam_dedup_stats_samtools_umitools/meta.yml cpan
subworkflows/nf-core/bam_markduplicates_picard/meta.yml cpan
subworkflows/nf-core/bam_rseqc/meta.yml cpan
subworkflows/nf-core/bam_sort_stats_samtools/meta.yml cpan
subworkflows/nf-core/bam_stats_samtools/meta.yml cpan
subworkflows/nf-core/bedgraph_bedclip_bedgraphtobigwig/meta.yml cpan
subworkflows/nf-core/fastq_align_hisat2/meta.yml cpan
subworkflows/nf-core/fastq_fastqc_umitools_fastp/meta.yml cpan
subworkflows/nf-core/fastq_fastqc_umitools_trimgalore/meta.yml cpan
subworkflows/nf-core/fastq_subsample_fq_salmon/meta.yml cpan
pyproject.toml pypi
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.github/workflows/ci.yml actions
  • actions/cache v3 composite
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  • nf-core/setup-nextflow v1 composite
.github/workflows/clean-up.yml actions
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.github/workflows/cloud_tests_full.yml actions
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.github/workflows/cloud_tests_small.yml actions
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modules/local/preparegenome/meta.yml cpan
modules/local/quant/meta.yml cpan
modules/local/samtools/flagstat/meta.yml cpan
modules/local/samtools/idxstats/meta.yml cpan
modules/local/samtools/index/meta.yml cpan
modules/local/samtools/sort/meta.yml cpan
modules/local/samtools/stats/meta.yml cpan
modules/local/convertspgxf2gxf/environment.yml conda
  • agat 1.0.0.*
modules/local/quant/environment.yml conda
  • salmon 1.10.1.*
modules/local/preparegenome/environment.yml pypi
modules/local/samtools/flagstat/environment.yml pypi
modules/local/samtools/idxstats/environment.yml pypi
modules/local/samtools/index/environment.yml pypi
modules/local/samtools/sort/environment.yml pypi
modules/local/samtools/stats/environment.yml pypi
modules/nf-core/bbmap/bbsplit/environment.yml pypi
modules/nf-core/cat/fastq/environment.yml pypi
modules/nf-core/custom/dumpsoftwareversions/environment.yml pypi
modules/nf-core/custom/getchromsizes/environment.yml pypi
modules/nf-core/fastp/environment.yml pypi
modules/nf-core/fastqc/environment.yml pypi
modules/nf-core/fq/subsample/environment.yml pypi
modules/nf-core/gffread/environment.yml pypi
modules/nf-core/gunzip/environment.yml pypi
modules/nf-core/hisat2/align/environment.yml pypi
modules/nf-core/hisat2/build/environment.yml pypi
modules/nf-core/hisat2/extractsplicesites/environment.yml pypi
modules/nf-core/picard/markduplicates/environment.yml pypi
modules/nf-core/preseq/lcextrap/environment.yml pypi
modules/nf-core/qualimap/rnaseq/environment.yml pypi
modules/nf-core/rsem/calculateexpression/environment.yml pypi
modules/nf-core/rsem/preparereference/environment.yml pypi
modules/nf-core/rseqc/bamstat/environment.yml pypi
modules/nf-core/rseqc/inferexperiment/environment.yml pypi
modules/nf-core/rseqc/innerdistance/environment.yml pypi
modules/nf-core/rseqc/junctionannotation/environment.yml pypi