RNA-seq pipeline parameters

Methods and parameters for the out-of-the-box RNA-seq pipeline (nf-core/rnaseq) offered in Pluto

Overview

On all plans in Pluto, scientists are able to run a streamlined, community-validated RNA-seq pipeline, nf-core/rnaseq. Written with Nextflow and available as an open source repository on Github, this pipeline is actively maintained and widely considered the industry-standard for analyzing RNA-seq data.

To run the pipeline, you'll upload a sample annotation table and raw FASTQ files. From there, you'll be prompted to select 3 different parameters:

• Genome build - Pluto will show you available genome builds based on the organism you selected when creating the experiment (e.g. GRCh38 / hg38 for human samples)
• Strandedness - If you know the strandedness of your experiment, select forward, reverse, or unstranded. If you don't know, simply select auto and the pipeline will infer strandedness.
• Library type - If your experiment generated both R1 and R2 FASTQ files, select paired-end, otherwise select single-end.

The pipeline outputs:

• Dynamic methods section with references
• MultiQC report
• BAM/BAI files (and additional pipeline intermediates depending on your plan tier)
• Raw count matrix, which you'll then use for flexible downstream analysis on Pluto's canvas

Citable methods

Raw FASTQ files were processed using the nf-core/rnaseq pipeline (v3.7) (1, 2). Adapter sequences were removed with Trim Galore (3). Reads were aligned with STAR (4) to GRCh38/hg19 (human) or GRCm38/mm10 (mouse) and quantified to gene counts using RSEM (5).

References

Other parameters

Besides the 3 user-editable parameters above, the nf-core/rnaseq pipeline has numerous other parameters. View the full list on the nf-core website for more details.

In Pluto's Starter tiers, these parameters are set to reasonable and robust default values that have been shown to produce reliable results across a wide variety of experiments. Below is the full list of parameters with default values (you can also download the parameters as JSON):

{
    "remove_ribo_rna": false,
    "custom_config_base": "https://raw.githubusercontent.com/nf-core/configs/master",
    "skip_deseq2_qc": true,
    "gencode": false,
    "umitools_dedup_stats": false,
    "plaintext_email": false,
    "gene_bed": "",
    "save_reference": false,
    "skip_markduplicates": false,
    "ribo_database_manifest": "/.nextflow/assets/pluto-biosciences/pluto-pipelines-rnaseq/assets/rrna-db-defaults.txt",
    "monochrome_logs": false,
    "aligner": "star_rsem",
    "max_cpus": 32,
    "featurecounts_group_type": "gene_biotype",
    "save_bbsplit_reads": false,
    "skip_multiqc": false,
    "skip_preseq": true,
    "skip_dupradar": false,
    "save_align_intermeds": true,
    "gtf": <organism-specific GTF>,
    "max_multiqc_email_size": "25.MB",
    "use_spot": true,
    "max_time": "240.h",
    "save_trimmed": false,
    "min_trimmed_reads": 10000,
    "pipeline_type": "experiment",
    "skip_fastqc": false,
    "deseq2_vst": true,
    "umitools_extract_method": "string",
    "validate_params": true,
    "config_profile_contact": "Hatem Nawar @hnawar",
    "bam_csi_index": false,
    "with_umi": false,
    "skip_qc": false,
    "version": false,
    "trimmer": "trimgalore",
    "publish_dir_mode": "copy",
    "input": <user-created sample annotation file>,
    "skip_bigwig": false,
    "strandedness": "auto",
    "igenomes_base": "s3://ngi-igenomes/igenomes",
    "stringtie_ignore_gtf": false,
    "config_profile_url": "https://cloud.google.com/batch",
    "use_private_ip": true,
    "skip_umi_extract": false,
    "google_bucket": false,
    "save_unaligned": false,
    "featurecounts_feature_type": "exon",
    "multiqc_title": <experiment ID>,
    "custom_config_version": "master",
    "max_memory": "128.GB",
    "boot_disk": "100 GB",
    "fasta": <organism-specific fasta>,
    "hisat2_build_memory": "200.GB",
    "skip_rseqc": false,
    "save_non_ribo_reads": false,
    "skip_alignment": false,
    "skip_qualimap": false,
    "star_index": "",
    "star_ignore_sjdbgtf": false,
    "skip_stringtie": true,
    "gtf_extra_attributes": "gene_name",
    "save_merged_fastq": true,
    "rsem_index": <organism-specific index>,
    "save_umi_intermeds": false,
    "schema_ignore_params": "genomes",
    "config_profile_description": "Google Cloud Batch API Profile",
    "min_mapped_reads": 5,
    "rseqc_modules": "bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication",
    "experiment_id": <experiment ID>,
    "google_debug": false,
    "gtf_group_features": "gene_id",
    "skip_trimming": false,
    "igenomes_ignore": false,
    "skip_pseudo_alignment": false,
    "outdir": <out directory>,
    "skip_bbsplit": true,
    "genome": <user-selected genome build>,
    "help": false,
    "show_hidden_params": false,
    "test_data_base": "https://raw.githubusercontent.com/nf-core/test-datasets/rnaseq3",
    "google_preemptible": true,
    "location": <GCP region>,
    "umitools_grouping_method": "directional",
    "google_zone": <GCP zone>,
    "skip_biotype_qc": true
}