Microbial RNA-seq

Summary of methods

• Index generated with RSEM
• Filter and trim reads with fastp
• Quality checking of reads with FastQC
• Reads mapped to reference and quantified with RSEM
• Alignment post-processing and QC with Picard
• Aggregation of QC tables using MultiQC

Parameters

Required parameters:

• --pubdir

  • Default: /<PATH>
  • Description: The directory that the saved outputs will be stored.

• -w

  • Default: /<PATH>
  • The directory that all intermediary files and nextflow processes utilize. This directory can become quite large. This should be a location on scratch space or other directory with ample storage.

• sample_folder

  • Default: /<PATH>
  • The path to the folder that contains all the samples to be run by the pipeline. The files in this path can also be symbolic links.

• strandedness

  • Default: NA
  • Supported options are reverse_stranded, forward_stranded, non_stranded

• --fasta

  • Default: /<PATH>
  • Path to the reference genome in FASTA format

• --gff

  • Default: /<PATH>
  • Path to the annotation for the reference genome in GFF3 format

• --read_type

  • Default: PE
  • Comment: Type of reads: paired end (PE) or single end (SE).

fastp filtering paramenters:

• --quality_phred

  • Default: 15
  • Quality score threshold.

• --unqualified_perc

  • Default: 40
  • Percent threhold of unqualified bases to pass reads.

Pipeline Default Outputs

Naming Convention	Description
microbial_rnaseq_report.html	Nextflow autogenerated report
trace.txt	Nextflow trace of processes
multiqc/	MultiQC report summarizing quality metrics across all samples in the run
${sampleID}/bam/${sampleID}.genome.bam	RSEM-generated alignment of reads to the reference genome
${sampleID}/bam/${sampleID}.transcript.bam	RSEM-generated alignment of reads to the reference transcriptome
${sampleID}/${sampleID}.genes.results	RSEM-generated quantification of gene-level count abundances
${sampleID}/${sampleID}.isoforms.results	RSEM-generated quantification of transcript-level count abundances

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Last update: January 13, 2024