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rnaseq.md

118 lines · bio-research/skills/nextflow-development/references/pipelines/rnaseq.md at 2574e29

1# nf-core/rnaseq

3**Version:** 3.22.2

5**Official Documentation:** https://nf-co.re/rnaseq/3.22.2/

6**GitHub:** https://github.com/nf-core/rnaseq

8> **Note:** When updating to a new version, check the [releases page](https://github.com/nf-core/rnaseq/releases) for breaking changes and update the version in commands below.

nf-core/rnaseq

Version: 3.22.2

Official Documentation: https://nf-co.re/rnaseq/3.22.2/ GitHub: https://github.com/nf-core/rnaseq

Note: When updating to a new version, check the releases page for breaking changes and update the version in commands below.

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10## Contents

11- [Test command](#test-command)

12- [Samplesheet format](#samplesheet-format)

13- [Parameters](#parameters)

14- [Output files](#output-files)

15- [Downstream analysis](#downstream-analysis)

Test command
Samplesheet format
Parameters
Output files
Downstream analysis

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17## Test command

19```bash

20nextflow run nf-core/rnaseq -r 3.22.2 -profile test,docker --outdir test_rnaseq

21```

23Expected: ~15 min, creates `multiqc/multiqc_report.html`.

Test command

nextflow run nf-core/rnaseq -r 3.22.2 -profile test,docker --outdir test_rnaseq

Expected: ~15 min, creates multiqc/multiqc_report.html.

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25## Samplesheet format

27```csv

28sample,fastq_1,fastq_2,strandedness

29CONTROL_REP1,/path/to/ctrl1_R1.fq.gz,/path/to/ctrl1_R2.fq.gz,auto

30CONTROL_REP2,/path/to/ctrl2_R1.fq.gz,/path/to/ctrl2_R2.fq.gz,auto

31TREATMENT_REP1,/path/to/treat1_R1.fq.gz,/path/to/treat1_R2.fq.gz,auto

32```

34| Column | Required | Values |

35|--------|----------|--------|

36| sample | Yes | Alphanumeric, underscores allowed |

37| fastq_1 | Yes | Absolute path to R1 |

38| fastq_2 | No | Absolute path to R2 (empty for single-end) |

39| strandedness | Yes | `auto`, `forward`, `reverse`, `unstranded` |

41**Strandedness guide:**

42- `auto`: Inferred from data (recommended)

43- `forward`: TruSeq Stranded, dUTP protocols

44- `reverse`: Ligation-based protocols

45- `unstranded`: Non-stranded protocols

Samplesheet format

sample,fastq_1,fastq_2,strandedness
CONTROL_REP1,/path/to/ctrl1_R1.fq.gz,/path/to/ctrl1_R2.fq.gz,auto
CONTROL_REP2,/path/to/ctrl2_R1.fq.gz,/path/to/ctrl2_R2.fq.gz,auto
TREATMENT_REP1,/path/to/treat1_R1.fq.gz,/path/to/treat1_R2.fq.gz,auto

Column	Required	Values
sample	Yes	Alphanumeric, underscores allowed
fastq_1	Yes	Absolute path to R1
fastq_2	No	Absolute path to R2 (empty for single-end)
strandedness	Yes	`auto`, `forward`, `reverse`, `unstranded`

Strandedness guide:

auto: Inferred from data (recommended)
forward: TruSeq Stranded, dUTP protocols
reverse: Ligation-based protocols
unstranded: Non-stranded protocols

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47## Parameters

Parameters

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49### Minimal run

50```bash

51nextflow run nf-core/rnaseq -r 3.22.2 -profile docker \

52 --input samplesheet.csv --outdir results --genome GRCh38

53```

Minimal run

nextflow run nf-core/rnaseq -r 3.22.2 -profile docker \
    --input samplesheet.csv --outdir results --genome GRCh38

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55### Common parameters

57| Parameter | Default | Description |

58|-----------|---------|-------------|

59| `--aligner` | `star_salmon` | Options: `star_salmon`, `star_rsem`, `hisat2` |

60| `--genome` | - | `GRCh38`, `GRCh37`, `mm10`, `BDGP6` |

61| `--pseudo_aligner` | - | Set to `salmon` for pseudo-alignment only |

62| `--skip_trimming` | false | Skip adapter trimming |

63| `--skip_alignment` | false | Pseudo-alignment only |

Common parameters

Parameter	Default	Description
`--aligner`	`star_salmon`	Options: `star_salmon`, `star_rsem`, `hisat2`
`--genome`	-	`GRCh38`, `GRCh37`, `mm10`, `BDGP6`
`--pseudo_aligner`	-	Set to `salmon` for pseudo-alignment only
`--skip_trimming`	false	Skip adapter trimming
`--skip_alignment`	false	Pseudo-alignment only

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65### Custom reference

66```bash

67--fasta /path/to/genome.fa \

68--gtf /path/to/annotation.gtf \

69--star_index /path/to/star/ # Optional, builds if absent

70```

Custom reference

--fasta /path/to/genome.fa \
--gtf /path/to/annotation.gtf \
--star_index /path/to/star/  # Optional, builds if absent

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72## Output files

74```

75results/

76├── star_salmon/

77│ ├── salmon.merged.gene_counts.tsv # Raw counts for DESeq2

78│ ├── salmon.merged.gene_tpm.tsv # TPM values

79│ └── *.bam # Alignments

80├── multiqc/

81│ └── multiqc_report.html # QC summary

82└── pipeline_info/

83```

85**Key outputs:**

86- `salmon.merged.gene_counts.tsv`: Input for DESeq2/edgeR

87- `salmon.merged.gene_tpm.tsv`: Normalized expression

Output files

results/
├── star_salmon/
│   ├── salmon.merged.gene_counts.tsv    # Raw counts for DESeq2
│   ├── salmon.merged.gene_tpm.tsv       # TPM values
│   └── *.bam                            # Alignments
├── multiqc/
│   └── multiqc_report.html              # QC summary
└── pipeline_info/

Key outputs:

salmon.merged.gene_counts.tsv: Input for DESeq2/edgeR
salmon.merged.gene_tpm.tsv: Normalized expression

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89## Downstream analysis

91```r

92library(DESeq2)

93counts <- read.delim("salmon.merged.gene_counts.tsv", row.names=1)

94coldata <- data.frame(

95 condition = factor(c("control", "control", "treatment", "treatment"))

96)

97dds <- DESeqDataSetFromMatrix(

98 countData = round(counts),

99 colData = coldata,

100 design = ~ condition

101)

102dds <- DESeq(dds)

103res <- results(dds, contrast = c("condition", "treatment", "control"))

104```

105

Downstream analysis

library(DESeq2)
counts <- read.delim("salmon.merged.gene_counts.tsv", row.names=1)
coldata <- data.frame(
    condition = factor(c("control", "control", "treatment", "treatment"))
)
dds <- DESeqDataSetFromMatrix(
    countData = round(counts),
    colData = coldata,
    design = ~ condition
)
dds <- DESeq(dds)
res <- results(dds, contrast = c("condition", "treatment", "control"))

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106## Troubleshooting

107

108**STAR index fails**: Increase memory with `--max_memory '64.GB'` or provide pre-built `--star_index`.

109

110**Low alignment rate**: Verify genome matches species; check FastQC for adapter contamination.

111

112**Strandedness detection fails**: Specify explicitly with `--strandedness reverse`.

113

Troubleshooting

STAR index fails: Increase memory with --max_memory '64.GB' or provide pre-built --star_index.

Low alignment rate: Verify genome matches species; check FastQC for adapter contamination.

Strandedness detection fails: Specify explicitly with --strandedness reverse.

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114## More Information

115

116- **Full parameter list:** https://nf-co.re/rnaseq/3.22.2/parameters/

117- **Output documentation:** https://nf-co.re/rnaseq/3.22.2/docs/output/

118- **Usage documentation:** https://nf-co.re/rnaseq/3.22.2/docs/usage/

More Information

Full parameter list: https://nf-co.re/rnaseq/3.22.2/parameters/
Output documentation: https://nf-co.re/rnaseq/3.22.2/docs/output/
Usage documentation: https://nf-co.re/rnaseq/3.22.2/docs/usage/

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