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 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 |
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 |
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 |
FASTQ/TRIMMED_FASTQ |
FASTQ/BAM |
|
Picard tools |
A better duplication marking algorithm that handles all cases including clipped and gapped alignments. |
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 |
BAM/FASTQ |
TEXT |
|
Salmon |
Salmon is a tool for wicked-fast transcript quantification from RNA-seq data. |
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 |
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 |
BAM |
BED/BEDG/BIGWIG |
|
MACS |
Model-based Analysis of ChIP-Seq (MACS), for identifying transcript factor binding sites. |
macs |
macs |
BAM |
BED/BEDG/BIGWIG |
|
SciPhy |
Software for cyPhyRNA-Seq Data analysis |
scyphy |
piranha |
BAM |
BED/BEDG/BIGWIG |
|
Peaks |
Slinding window peak finding tool for quick assessment of peaks. UNPUBLISHED, recommended for initial scanning only |
peaks |
peaks |
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 |
BAM |
BED/BEDG/BIGWIG |