Available Workflows

FETCH -> Download from SRA

BASECALL -> Call bases from FAST5 or POD5

QC -> Quality Control of FASTQ/BAM files

TRIMMING -> Trim adaptor remnants

MAPPING -> Map reads to reference

DEDUP -> Deduplicate reads

COUNTING -> Count or quantify reads

TRACKS -> Generates trackdb.txt and BIGWIG files

PEAKS -> Call peaks for ChIP, RIP, CLIP and other

DE -> Differential Expression Analysis

DEU -> Differential Exon Usage Analysis

DAS -> Differential Alternative Splicing Analysis

DTU -> Differential Transcript Usage Analysis

CIRCS -> Circular RNA identification

PREPROCESSING

FETCH

Downloads files from SRA

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

SRAtools fasterq-dump

The fasterq-dump tool extracts data in FASTQ- or FASTA-format from SRA-accessions

sra

fasterq-dump

sra

SRA accession

FASTQ

BASECALL

Creates FASTQ files from ONT FAST5, Guppy needs to be installed locally

QUALITY CONTROL I

This workflow step can be run as preprocessing step if none of the processing workflows is defined in the config.json.

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

FASTQC (includes MULTIQC)

A quality control tool for high throughput sequence data.

fastqc

fastqc

fastqc

FASTQ/BAM

ZIP/HTML

PROCESSING

QUALITY CONTROL II

If any of the below listed processing steps is defined in the config.json, quality control will run for all generated output files if enabled.

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

FASTQC (includes MULTIQC)

A quality control tool for high throughput sequence data.

fastqc

fastqc

fastqc

FASTQ/BAM

ZIP/HTML

Trimming

Trims adaptor left-overs from fastq files

Mapping

Maps sequences to reference genomes or transcriptomes

DEDUP

Deduplicate reads by UMI or based on mapping position and CIGAR string

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

UMI-tools

UMI-tools contains tools for dealing with Unique Molecular Identifiers (UMIs)/Random Molecular Tags (RMTs) and single cell RNA-Seq cell barcodes.

umitools

umi_tools

umitools

FASTQ/TRIMMED_FASTQ

FASTQ/BAM

Picard tools

A better duplication marking algorithm that handles all cases including clipped and gapped alignments.

picard

picard

picard

BAM

BAM

POSTPROCESSING

Read-Counting and Quantification

Count (unique) mapped reads and how often they map to defined features

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

FeatureCounts

A software program developed for counting reads to genomic features such as genes, exons, promoters and genomic bins

countreads

featureCounts

featurecounts

BAM/FASTQ

TEXT

Salmon

Salmon is a tool for wicked-fast transcript quantification from RNA-seq data.

salmon

salmon

salmon

FASTQ/TRIMMED_FASTQ

TEXT

Differential Analyses

Includes DE, DEU, DAS and DTU

Tool

Analysis

Filtering

Normalization

Distribution

Testing

Significance

Results Table

further

SigTables

Clustering

further

Rmd

edgeR

Differential Gene Expression

filterByExpr()

TMM

NB

Fisher’s exact test

pValue, LFC

results, sorted-results

normalized

Sig, SigUP, SigDOWN

MDS-plot

BCV, QLDisp, MD(per comparison)

edgeR

Differential Exon Usage

filterByExpr()

TMM

NB

Fisher’s exact test

pValue, LFC

results

normalized

MDS-plot

BCV, QLDisp, MD(per comparison)

edgeR

Differential Alternative Splicing

filterByExpr()

TMM

NB

Simes, gene-level, exon-level

pValue, LFC

results(diffSpliceExonTest, Simes-Test, Gene-Test)

Sig, SigUP, SigDOWN

MDS-plot

BCV, QLDisp, MD(per comparison), topSpliceSimes-plots(per Gene)

DESeq2

Differential Gene Expression

RowSums >= 10

RLE

NB

Wald test

pValue, LFC

results

rld, vsd, results(per comparison)

Sig, SigUP, SigDOWN

PCA

Heatmaps, MA(per comparison), VST-and-log2

DEXSeq

Differential Exon Usage

RowSums >= 10

RLE

Cox-Reid

likelihood ratio test

DEXSeq

Differential Transcript Usage

dmFilter()

RLE

Cox-Reid

likelihood ratio test

pValue

results

DIEGO

Differential Alternative Splicing

Mann-Whitney U test

pValue

results

Sig

Dendrogram-plot

DRIMSeq

Differential Transcript Usage

dmFilter()

DM

pValue, LFC

results(transcript, genes)

Proportions-table, genewise precision

Sig, SigUP, SigDOWN (transcipt, gene)

FeatPerGene, precision, Pvalues (per comparison)

TRACKS

This workflow generates trackdb.txt files and bigwig files which can be used to create UCSC track hubs. However, bigwigs can of course be used for other genome viewers as well.

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

UCSC

Track hubs are web-accessible directories of genomic data that can be viewed on the UCSC Genome Browser

ucsc

ucsc

ucsc

BAM

BIGWIG/HUBS

PEAKS

Calls peaks from mapping data for ChIP, RIP, CLIP and other

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

Piranha

Piranha is a peak-caller for CLIP- and RIP-Seq data.

piranha

piranha

piranha

BAM

BED/BEDG/BIGWIG

MACS

Model-based Analysis of ChIP-Seq (MACS), for identifying transcript factor binding sites.

macs

macs

macs

BAM

BED/BEDG/BIGWIG

SciPhy

Software for cyPhyRNA-Seq Data analysis

scyphy

piranha

cyphyRNA-Seq

BAM

BED/BEDG/BIGWIG

Peaks

Slinding window peak finding tool for quick assessment of peaks. UNPUBLISHED, recommended for initial scanning only

peaks

peaks

ttp

BAM

BED/BEDG/BIGWIG

CIRCS

Find circular RNAs in mapping data, CIRI2 needs to be installed locally.

TOOL

DESCRIPTION

ENV

BIN

LINK

INPUT

OUTPUT

CIRI2

CIRI (circRNA identifier) is a novel chiastic clipping signal based algorithm,which can unbiasedly and accurately detect circRNAs from transcriptome data by employing multiple filtration strategies.

ciri2

$Path_to_CIRI2.pl

ciri2

BAM

BED/BEDG/BIGWIG