nf-core/rnaseq

nf-core pipelines

RNA-seq analysis with multiple aligner and quantification routes (STAR/RSEM, STAR/Salmon, Bowtie2/Salmon, HISAT2, Salmon pseudo-alignment, Kallisto). GitHub

Mermaid source

examples/rnaseq_metro.mmd

%%metro title: nf-core/rnaseq
%%metro logo: nf-core-rnaseq_logo_light.png | nf-core-rnaseq_logo_dark.png
%%metro style: dark
%%metro diamond_style: symmetric
%%metro file: fastq_in | FASTQ
%%metro file: report_final | HTML
%%metro file: report_quant | HTML
%%metro file: report_bowtie2 | HTML
%%metro line: star_rsem | Aligner: STAR, Quantification: RSEM | #0570b0
%%metro line: star_salmon | Aligner: STAR, Quantification: Salmon (default) | #2db572
%%metro line: bowtie2_salmon | Aligner: Bowtie2 (prokaryotic), Quantification: Salmon | #ff8c00
%%metro line: hisat2 | Aligner: HISAT2, Quantification: None | #f5c542
%%metro line: pseudo_salmon | Pseudo-aligner: Salmon, Quantification: Salmon | #e63946
%%metro line: pseudo_kallisto | Pseudo-aligner: Kallisto, Quantification: Kallisto | #7b2d3b
%%metro legend: bl
%%metro logo_scale: 0.85

%% Process mappings for live-progress mode (nf-metro serve / nf-metro check-mapping).
%% process_scope factors out the shared FQN prefix so each value is the tail
%% under NFCORE_RNASEQ:RNASEQ, matched literally with intermediate subworkflow
%% nesting tolerated.
%%metro process_scope: NFCORE_RNASEQ:RNASEQ

%% Pre-processing
%%metro process: cat_fastq          | CAT_FASTQ
%%metro process: fastqc_raw         | FASTQ_FASTQC_UMITOOLS_TRIMGALORE:FASTQC
%%metro process: fastqc_raw         | FASTQ_FASTQC_UMITOOLS_FASTP:FASTQC_RAW
%%metro process: umi_tools_extract  | UMITOOLS_EXTRACT
%%metro process: fastp              | FASTP
%%metro process: trimgalore         | TRIMGALORE
%%metro process: fastqc_trimmed     | FASTQ_FASTQC_UMITOOLS_FASTP:FASTQC_TRIM
%%metro process: bbsplit            | BBMAP_BBSPLIT
%%metro process: sortmerna          | FASTQ_REMOVE_RRNA:SORTMERNA
%%metro process: ribodetector       | RIBODETECTOR
%%metro process: bowtie2_rrna       | FASTQ_QC_TRIM_FILTER_SETSTRANDEDNESS:FASTQ_REMOVE_RRNA:BOWTIE2_ALIGN
%%metro process: bowtie2_rrna       | FASTQ_QC_TRIM_FILTER_SETSTRANDEDNESS:FASTQ_REMOVE_RRNA:BOWTIE2_ALIGN_PE
%%metro process: fastqc_filtered    | FASTQC_FILTERED
%%metro process: infer_strandedness | FASTQ_SUBSAMPLE_FQ_SALMON:SALMON_QUANT

%% Genome alignment & quantification
%%metro process: star               | ALIGN_STAR:STAR_ALIGN
%%metro process: star               | ALIGN_STAR:SENTIEON_STAR_ALIGN
%%metro process: star               | ALIGN_STAR:PARABRICKS_RNA_FQ2BAM
%%metro process: hisat2_align       | FASTQ_ALIGN_HISAT2:HISAT2_ALIGN
%%metro process: bowtie2_align      | ALIGN_BOWTIE2:BOWTIE2_ALIGN
%%metro process: bowtie2_align      | ALIGN_BOWTIE2:BOWTIE2_ALIGN_PE
%%metro process: umi_tools_dedup    | UMITOOLS_DEDUP
%%metro process: umi_tools_dedup    | UMICOLLAPSE
%%metro process: rsem               | RSEM_CALCULATEEXPRESSION
%%metro process: rsem               | SENTIEON_RSEMCALCULATEEXPRESSION
%%metro process: salmon_quant       | QUANTIFY_BAM_SALMON:SALMON_QUANT
%%metro process: tximport_ga        | QUANTIFY_BAM_SALMON:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:TXIMETA_TXIMPORT
%%metro process: tximport_ga        | QUANTIFY_RSEM:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:TXIMETA_TXIMPORT
%%metro process: summarized_exp_ga  | QUANTIFY_BAM_SALMON:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_GENE_UNIFIED
%%metro process: summarized_exp_ga  | QUANTIFY_BAM_SALMON:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_TRANSCRIPT_UNIFIED
%%metro process: summarized_exp_ga  | QUANTIFY_RSEM:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_GENE_UNIFIED
%%metro process: summarized_exp_ga  | QUANTIFY_RSEM:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_TRANSCRIPT_UNIFIED
%%metro process: multiqc_bowtie2    | MULTIQC_RNASEQ:MULTIQC

%% Pseudo-alignment & quantification
%%metro process: salmon_pseudo      | QUANTIFY_PSEUDO_ALIGNMENT:SALMON_QUANT
%%metro process: kallisto           | KALLISTO_QUANT
%%metro process: tximport_pa        | QUANTIFY_PSEUDO_ALIGNMENT:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:TXIMETA_TXIMPORT
%%metro process: summarized_exp_pa  | QUANTIFY_PSEUDO_ALIGNMENT:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_GENE_UNIFIED
%%metro process: summarized_exp_pa  | QUANTIFY_PSEUDO_ALIGNMENT:QUANT_TXIMPORT_SUMMARIZEDEXPERIMENT:SE_TRANSCRIPT_UNIFIED
%%metro process: multiqc_quant      | MULTIQC_RNASEQ:MULTIQC

%% Post-processing
%%metro process: picard             | PICARD_MARKDUPLICATES
%%metro process: bedtools           | BEDTOOLS_GENOMECOV_FW
%%metro process: bedtools           | BEDTOOLS_GENOMECOV_REV
%%metro process: bedtools           | BEDTOOLS_GENOMECOV_COMBINED
%%metro process: bedgraph           | UCSC_BEDGRAPHTOBIGWIG
%%metro process: stringtie          | STRINGTIE_STRINGTIE

%% Quality control & reporting
%%metro process: rseqc              | RSEQC_BAMSTAT
%%metro process: rseqc              | RSEQC_INNERDISTANCE
%%metro process: rseqc              | RSEQC_INFEREXPERIMENT
%%metro process: rseqc              | RSEQC_JUNCTIONANNOTATION
%%metro process: rseqc              | RSEQC_JUNCTIONSATURATION
%%metro process: rseqc              | RSEQC_READDISTRIBUTION
%%metro process: rseqc              | RSEQC_READDUPLICATION
%%metro process: rseqc              | RSEQC_TIN
%%metro process: preseq             | PRESEQ_LCEXTRAP
%%metro process: qualimap           | QUALIMAP_RNASEQ
%%metro process: dupradar           | DUPRADAR
%%metro process: featurecounts      | SUBREAD_FEATURECOUNTS
%%metro process: deseq2_pca         | DESEQ2_QC_BAM_SALMON
%%metro process: deseq2_pca         | DESEQ2_QC_RSEM
%%metro process: deseq2_pca         | DESEQ2_QC_PSEUDO
%%metro process: kraken2            | KRAKEN2
%%metro process: sylph              | SYLPH_PROFILE
%%metro process: sylph              | SYLPHTAX_TAXPROF
%%metro process: multiqc_final      | MULTIQC_RNASEQ:MULTIQC

graph LR
    subgraph preprocessing [Pre-processing]
        %%metro exit: right | star_salmon, star_rsem, hisat2, bowtie2_salmon
        %%metro exit: bottom | pseudo_salmon, pseudo_kallisto
        fastq_in[ ]
        cat_fastq[cat FASTQ]
        fastqc_raw[FastQC]
        umi_tools_extract[UMI-tools Extract]
        fastp[fastp]
        trimgalore[Trim Galore!]
        fastqc_trimmed[FastQC]
        bbsplit[BBSplit]
        sortmerna[SortMeRNA]
        ribodetector[RiboDetector]
        bowtie2_rrna[Bowtie2]
        fastqc_filtered[FastQC]
        infer_strandedness[Infer Strand.]

        fastq_in -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| cat_fastq
        cat_fastq -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_raw
        fastqc_raw -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| umi_tools_extract

        umi_tools_extract -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastp
        umi_tools_extract -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| trimgalore
        fastp -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_trimmed
        trimgalore -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_trimmed

        fastqc_trimmed -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| bbsplit
        bbsplit -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| sortmerna
        bbsplit -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| ribodetector
        bbsplit -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| bowtie2_rrna
        sortmerna -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_filtered
        ribodetector -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_filtered
        bowtie2_rrna -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| fastqc_filtered
        fastqc_filtered -->|pseudo_salmon,pseudo_kallisto,star_salmon,star_rsem,hisat2,bowtie2_salmon| infer_strandedness
    end

    subgraph genome_align [Genome alignment & quantification]
        %%metro entry: left | star_salmon, star_rsem, hisat2, bowtie2_salmon
        %%metro exit: right | star_salmon, star_rsem
        %%metro exit: right | hisat2
        star[STAR]
        hisat2_align[HISAT2]
        bowtie2_align[Bowtie2]
        rsem[RSEM]
        salmon_quant[Salmon]
        umi_tools_dedup[UMI-tools Dedup]
        tximport_ga[tximport]
        summarized_exp_ga[Sum. Exp.]
        multiqc_bowtie2[MultiQC]
        report_bowtie2[ ]
        _h1[hidden]
        _h2[hidden]
        _h3[hidden]

        star -->|star_rsem,star_salmon| umi_tools_dedup
        hisat2_align -->|hisat2| umi_tools_dedup
        bowtie2_align -->|bowtie2_salmon| umi_tools_dedup
        umi_tools_dedup -->|star_rsem| rsem
        umi_tools_dedup -->|star_salmon,bowtie2_salmon| salmon_quant
        umi_tools_dedup -->|hisat2| _h1
        _h1 -->|hisat2| _h2
        _h2 -->|hisat2| _h3
        salmon_quant -->|star_salmon,bowtie2_salmon| tximport_ga
        rsem -->|star_rsem| tximport_ga
        tximport_ga -->|star_salmon,star_rsem,bowtie2_salmon| summarized_exp_ga
        summarized_exp_ga -->|bowtie2_salmon| multiqc_bowtie2
        multiqc_bowtie2 -->|bowtie2_salmon| report_bowtie2
    end

    subgraph pseudo_align [Pseudo-alignment & quantification]
        %%metro entry: left | pseudo_salmon, pseudo_kallisto
        salmon_pseudo[Salmon]
        kallisto[Kallisto]
        tximport_pa[tximport]
        summarized_exp_pa[Sum. Exp.]
        multiqc_quant[MultiQC]
        report_quant[ ]

        salmon_pseudo -->|pseudo_salmon| tximport_pa
        kallisto -->|pseudo_kallisto| tximport_pa
        tximport_pa -->|pseudo_salmon,pseudo_kallisto| summarized_exp_pa
        summarized_exp_pa -->|pseudo_salmon,pseudo_kallisto| multiqc_quant
        multiqc_quant -->|pseudo_salmon,pseudo_kallisto| report_quant
    end

    subgraph postprocessing [Post-processing]
        %%metro direction: TB
        %%metro entry: left | star_salmon, star_rsem, hisat2
        %%metro exit: bottom | star_salmon, star_rsem, hisat2
        picard[Picard]
        bedtools[BEDTools]
        bedgraph[bedGraphToBigWig]
        stringtie[StringTie]

        picard -->|star_salmon,star_rsem,hisat2| bedtools
        bedtools -->|star_salmon,star_rsem,hisat2| bedgraph
        bedgraph -->|star_salmon,star_rsem,hisat2| stringtie
    end

    subgraph qc_report [Quality control & reporting]
        %%metro direction: RL
        %%metro entry: top | star_salmon, star_rsem, hisat2
        rseqc[RSeQC]
        preseq[Preseq]
        qualimap[Qualimap]
        dupradar[dupRadar]
        featurecounts[featureCounts]
        deseq2_pca[DESeq2 PCA]
        kraken2[Kraken2/Bracken]
        sylph[Sylph]
        multiqc_final[MultiQC]
        report_final[ ]

        rseqc -->|star_salmon,star_rsem,hisat2| preseq
        preseq -->|star_salmon,star_rsem,hisat2| qualimap
        qualimap -->|star_salmon,star_rsem,hisat2| dupradar
        dupradar -->|star_salmon,star_rsem,hisat2| featurecounts
        featurecounts -->|star_salmon,star_rsem,hisat2| deseq2_pca
        deseq2_pca -->|star_salmon,star_rsem,hisat2| kraken2
        deseq2_pca -->|star_salmon,star_rsem,hisat2| sylph
        kraken2 -->|star_salmon,star_rsem,hisat2| multiqc_final
        sylph -->|star_salmon,star_rsem,hisat2| multiqc_final
        multiqc_final -->|star_salmon,star_rsem,hisat2| report_final
    end

    %% Inter-section edges
    infer_strandedness -->|star_salmon,star_rsem| star
    infer_strandedness -->|hisat2| hisat2_align
    infer_strandedness -->|bowtie2_salmon| bowtie2_align
    infer_strandedness -->|pseudo_salmon| salmon_pseudo
    infer_strandedness -->|pseudo_kallisto| kallisto
    summarized_exp_ga -->|star_salmon,star_rsem| picard
    _h3 -->|hisat2| picard
    stringtie -->|star_salmon,star_rsem,hisat2| rseqc

CLI command

nf-metro render examples/rnaseq_metro.mmd -o rnaseq_metro.svg

Rendered map