RNA Sequencing Pipeline

A Nextflow pipeline to perform quality control, alignment, and quantification of RNA sequencing data.

The pipeline was created to run on the ETH Euler cluster and it relies on the server's genome files. Thus, the pipeline needs to be adapted before running it in a different HPC cluster.

Pipeline steps

1. FastQC
2. FastQ Screen
3. Trim Galore
4. FastQC
5. STAR or HISAT2
6. Samtools sort
7. Samtools index
8. featureCounts
9. MultiQC

Required parameters

Path to the folder where the FASTQ files are located.

--input /cluster/work/nme/data/josousa/project/fastq/*fastq.gz

Output directory where the files will be saved.

--outdir /cluster/work/nme/data/josousa/project

Input optional parameters

• Option to force the pipeline to assign input as single-end.

  --single_end

  By default, the pipeline detects whether the input files are single-end or paired-end.

• Option to select RNA-Seq library strandness. This will only affect quantification.

  --strandness 'smartseq2' # Default (same as 'unstranded')
  --strandness 'forward'
  --strandness 'reverse'
  --strandness 'unstranded'

  This option will only affect quantification.

Genomes

• Reference genome used for alignment.

  --genome

  Available genomes:

      Mus_musculus_GRCm39 # Default
      Mus_musculus_GRCm38_p6
      Homo_sapiens_GRCh38_p14
      Rattus_norvegicus_mRatBN7_2
      Bos_taurus_ARS-UCD1_2
      Bos_taurus_ARS-UCD1_3
      Caenorhabditis_elegans_WBcel235
      Callithrix_jacchus_mCalJac1_pat_X
      Capra_hircus_ARS1
      Capreolus_capreolus_GCA_951849835_1
      Drosophila_melanogaster_BDGP6_46
      Escherichia_coli_ASM160652v1
      Macaca_fascicularis_Macaca_fascicularis_6_0
      Macaca_mulatta_Mmul_10
      Monodelphis_domestica_ASM229v1
      Pan_troglodytes_Pan_tro_3_0
      Saccharomyces_cerevisiae_R64-1-1
      Sus_scrofa_Sscrofa11_1

• Option to use a custom genome for alignment by providing an absolute path to a custom genome file.

  --custom_genome_file '/cluster/work/nme/data/josousa/project/genome/GRCm39.genome'

  Example of a genome file:

  name           GRCm39
  species        Mouse
  star           /cluster/work/nme/genomes/Mus_musculus/Ensembl/GRCm39/Sequence/STARIndex/
  hisat2         /cluster/work/nme/genomes/Mus_musculus/Ensembl/GRCm39/Sequence/Hisat2Index/genome
  hisat2_splices /cluster/work/nme/genomes/Mus_musculus/Ensembl/GRCm39/Sequence/Hisat2Index/splice_sites.txt
  gtf            /cluster/work/nme/genomes/Mus_musculus/Ensembl/GRCm39/Annotation/Genes/genes.gtf

Aligner options

• Option to choose the aligner.

  --aligner 'star' # Default
  --aligner 'hisat2'

HISAT2 parameters

• Option to choose no soft-clipping.

  --hisat2_no_softclip Default: true

• Option to suppress unpaired alignments for paired reads

  --hisat2_no_mixed Default: true

• Option to suppress discordant alignments for paired reads.

  --hisat2_no_discordant Default: true

FastQ Screen optional parameters

• Option to provide a custom FastQ Screen config file.

  --fastq_screen_conf '/cluster/work/nme/software/config/fastq_screen.conf' # Default

• Option to pass the flag --bisulfite to FastQ Screen.

  --bisulfite Default: false

featureCounts optional parameters

• Option to only count read pairs that have both ends aligned.

  --featurecounts_B_flag Default: true

• Option to not count read pairs that have their two ends mapping to different chromosomes or mapping to same chromosome but on different strands.

  --featurecounts_C_flag Default: true

Skipping options

• Option to skip FastQC, TrimGalore, and FastQ Screen. The first step of the pipeline will be the Bismark alignment. --skip_qc

• Option to skip FastQ Screen. --skip_fastq_screen

• Option to skip quantification. --skip_quantification

Extra arguments

• Option to add extra arguments to FastQC. --fastqc_args

• Option to add extra arguments to FastQ Screen. --fastq_screen_args

• Option to add extra arguments to Trim Galore. --trim_galore_args

• Option to add extra arguments to the STAR aligner. --star_align_args

• Option to add extra arguments to the HISAT2 aligner. --hisat2_align_args

• Option to add extra arguments to Samtools sort. --samtools_sort_args

• Option to add extra arguments to Samtools index. --samtools_index_args

• Option to add extra arguments to featureCounts. --featurecounts_args

• Option to add extra arguments to MultiQC. --multiqc_args

Acknowledgements

This pipeline was adapted from the Nextflow pipelines created by the Babraham Institute Bioinformatics Group and from the nf-core pipelines. We thank all the contributors for both projects. We also thank the Nextflow community and the nf-core community for all the help and support.