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atacseq.md

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

1# nf-core/atacseq

3**Version:** 2.1.2

5**Official Documentation:** https://nf-co.re/atacseq/2.1.2/

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

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

nf-core/atacseq

Version: 2.1.2

Official Documentation: https://nf-co.re/atacseq/2.1.2/ GitHub: https://github.com/nf-core/atacseq

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- [Quality metrics](#quality-metrics)

Test command
Samplesheet format
Parameters
Output files
Quality metrics

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17## Test command

19```bash

20nextflow run nf-core/atacseq -r 2.1.2 -profile test,docker --outdir test_atacseq

21```

23Expected: ~15 min, creates peaks and BigWig tracks.

Test command

nextflow run nf-core/atacseq -r 2.1.2 -profile test,docker --outdir test_atacseq

Expected: ~15 min, creates peaks and BigWig tracks.

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25## Samplesheet format

27```csv

28sample,fastq_1,fastq_2,replicate

29CONTROL,/path/to/ctrl_rep1_R1.fq.gz,/path/to/ctrl_rep1_R2.fq.gz,1

30CONTROL,/path/to/ctrl_rep2_R1.fq.gz,/path/to/ctrl_rep2_R2.fq.gz,2

31TREATMENT,/path/to/treat_rep1_R1.fq.gz,/path/to/treat_rep1_R2.fq.gz,1

32TREATMENT,/path/to/treat_rep2_R1.fq.gz,/path/to/treat_rep2_R2.fq.gz,2

33```

35| Column | Required | Description |

36|--------|----------|-------------|

37| sample | Yes | Condition/group identifier |

38| fastq_1 | Yes | Absolute path to R1 |

39| fastq_2 | Yes | Absolute path to R2 (paired-end required) |

40| replicate | Yes | Replicate number (integer) |

Samplesheet format

sample,fastq_1,fastq_2,replicate
CONTROL,/path/to/ctrl_rep1_R1.fq.gz,/path/to/ctrl_rep1_R2.fq.gz,1
CONTROL,/path/to/ctrl_rep2_R1.fq.gz,/path/to/ctrl_rep2_R2.fq.gz,2
TREATMENT,/path/to/treat_rep1_R1.fq.gz,/path/to/treat_rep1_R2.fq.gz,1
TREATMENT,/path/to/treat_rep2_R1.fq.gz,/path/to/treat_rep2_R2.fq.gz,2

Column	Required	Description
sample	Yes	Condition/group identifier
fastq_1	Yes	Absolute path to R1
fastq_2	Yes	Absolute path to R2 (paired-end required)
replicate	Yes	Replicate number (integer)

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42### Design file for differential analysis

43```csv

44sample,condition

45CONTROL,control

46TREATMENT,treatment

47```

49Use with `--deseq2_design design.csv`.

Design file for differential analysis

sample,condition
CONTROL,control
TREATMENT,treatment

Use with --deseq2_design design.csv.

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51## Parameters

Parameters

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53### Minimal run

54```bash

55nextflow run nf-core/atacseq -r 2.1.2 -profile docker \

56 --input samplesheet.csv --outdir results --genome GRCh38 --read_length 50

57```

Minimal run

nextflow run nf-core/atacseq -r 2.1.2 -profile docker \
    --input samplesheet.csv --outdir results --genome GRCh38 --read_length 50

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59### Common parameters

61| Parameter | Default | Description |

62|-----------|---------|-------------|

63| `--genome` | - | `GRCh38`, `GRCh37`, `mm10` |

64| `--read_length` | 50 | Read length for MACS2 optimization |

65| `--narrow_peak` | true | Narrow peaks (false for broad) |

66| `--mito_name` | chrM | Mitochondrial chromosome name |

67| `--keep_mito` | false | Keep mitochondrial reads |

68| `--min_reps_consensus` | 1 | Min replicates for consensus peaks |

Common parameters

Parameter	Default	Description
`--genome`	-	`GRCh38`, `GRCh37`, `mm10`
`--read_length`	50	Read length for MACS2 optimization
`--narrow_peak`	true	Narrow peaks (false for broad)
`--mito_name`	chrM	Mitochondrial chromosome name
`--keep_mito`	false	Keep mitochondrial reads
`--min_reps_consensus`	1	Min replicates for consensus peaks

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70### Differential accessibility

71```bash

72--deseq2_design design.csv

73```

Differential accessibility

--deseq2_design design.csv

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75## Output files

77```

78results/

79├── bwa/mergedLibrary/

80│ ├── *.mLb.mkD.sorted.bam # Filtered, deduplicated alignments

81│ └── bigwig/

82│ └── *.bigWig # Coverage tracks

83├── macs2/narrowPeak/

84│ ├── *.narrowPeak # Peak calls

85│ └── consensus/

86│ └── consensus_peaks.bed # Merged peaks across replicates

87├── deeptools/

88│ ├── plotFingerprint/ # Library complexity

89│ └── plotProfile/ # TSS enrichment

90├── deseq2/ # If --deseq2_design provided

91└── multiqc/

92```

94**Key outputs:**

95- `*.mLb.mkD.sorted.bam`: Analysis-ready alignments

96- `*.narrowPeak`: MACS2 peak calls (BED format)

97- `consensus_peaks.bed`: Consensus peaks across replicates

98- `*.bigWig`: Genome browser tracks

Output files

results/
├── bwa/mergedLibrary/
│   ├── *.mLb.mkD.sorted.bam     # Filtered, deduplicated alignments
│   └── bigwig/
│       └── *.bigWig             # Coverage tracks
├── macs2/narrowPeak/
│   ├── *.narrowPeak             # Peak calls
│   └── consensus/
│       └── consensus_peaks.bed  # Merged peaks across replicates
├── deeptools/
│   ├── plotFingerprint/         # Library complexity
│   └── plotProfile/             # TSS enrichment
├── deseq2/                      # If --deseq2_design provided
└── multiqc/

Key outputs:

*.mLb.mkD.sorted.bam: Analysis-ready alignments
*.narrowPeak: MACS2 peak calls (BED format)
consensus_peaks.bed: Consensus peaks across replicates
*.bigWig: Genome browser tracks

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100## Quality metrics

101

103|--------|------|------------|------|

104| Mapped reads | >80% | 60-80% | <60% |

105| Mitochondrial | <20% | 20-40% | >40% |

106| Duplicates | <30% | 30-50% | >50% |

107| FRiP | >30% | 15-30% | <15% |

108| TSS enrichment | >6 | 4-6 | <4 |

109

110**Fragment size**: Should show nucleosomal periodicity (~50bp nucleosome-free, ~200bp mono-nucleosome).

111

Quality metrics

Metric	Good	Acceptable	Poor
Mapped reads	>80%	60-80%	<60%
Mitochondrial	<20%	20-40%	>40%
Duplicates	<30%	30-50%	>50%
FRiP	>30%	15-30%	<15%
TSS enrichment	>6	4-6	<4

Fragment size: Should show nucleosomal periodicity (~50bp nucleosome-free, ~200bp mono-nucleosome).

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112## Downstream analysis

113

114```r

115library(ChIPseeker)

116library(GenomicRanges)

117peaks <- import("consensus_peaks.bed")

118peakAnno <- annotatePeak(peaks, TxDb = TxDb.Hsapiens.UCSC.hg38.knownGene)

119```

120

121**Motif analysis:**

122```bash

123findMotifsGenome.pl consensus_peaks.bed hg38 motifs/ -size 200

124```

125

Downstream analysis

library(ChIPseeker)
library(GenomicRanges)
peaks <- import("consensus_peaks.bed")
peakAnno <- annotatePeak(peaks, TxDb = TxDb.Hsapiens.UCSC.hg38.knownGene)

Motif analysis:

findMotifsGenome.pl consensus_peaks.bed hg38 motifs/ -size 200

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126## Troubleshooting

127

128**Low FRiP**: Check library complexity in `plotFingerprint/`. May indicate over-transposition.

129

130**Few peaks**: Lower threshold with `--macs_qvalue 0.1` or use `--narrow_peak false` for broader peaks.

131

132**High duplicates**: Normal for low-input; pipeline removes by default.

133

Troubleshooting

Low FRiP: Check library complexity in plotFingerprint/. May indicate over-transposition.

Few peaks: Lower threshold with --macs_qvalue 0.1 or use --narrow_peak false for broader peaks.

High duplicates: Normal for low-input; pipeline removes by default.

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134## More Information

135

136- **Full parameter list:** https://nf-co.re/atacseq/2.1.2/parameters/

137- **Output documentation:** https://nf-co.re/atacseq/2.1.2/docs/output/

138- **Usage documentation:** https://nf-co.re/atacseq/2.1.2/docs/usage/

More Information

Full parameter list: https://nf-co.re/atacseq/2.1.2/parameters/
Output documentation: https://nf-co.re/atacseq/2.1.2/docs/output/
Usage documentation: https://nf-co.re/atacseq/2.1.2/docs/usage/

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