nf-core/rnaseq v2.0 - Titanium Tiger

[2.0] - 2020-11-12

Major enhancements

• Pipeline has been re-implemented in Nextflow DSL2
• All software containers are now exclusively obtained from Biocontainers
• Added a separate workflow to download FastQ files via SRA, ENA or GEO ids and to auto-create the input samplesheet (ENA FTP; see --public_data_ids parameter)
• Added and refined a Groovy lib/ of functions that include the automatic rendering of parameters defined in the JSON schema for the help and summary log information
• Replace edgeR with DESeq2 for the generation of PCA and heatmaps (also included in the MultiQC report)
• Creation of bigWig coverage files using BEDTools and bedGraphToBigWig
• [#70] - Added new genome mapping and quantification route with RSEM via the --aligner star_rsem parameter
• [#72] - Samples skipped due to low alignment reported in the MultiQC report
• [#73, #435] - UMI barcode support
• [#91] - Ability to concatenate multiple runs of the same samples via the input samplesheet
• [#123] - The primary input for the pipeline has changed from --reads glob to samplesheet --input. See usage docs.
• [#197] - Samples failing strand-specificity checks reported in the MultiQC report
• [#227] - Removal of ribosomal RNA via SortMeRNA
• [#419] - Add --additional_fasta parameter to provide ERCC spike-ins, transgenes such as GFP or CAR-T as additional sequences to align to

Other enhancements & fixes

• Updated pipeline template to nf-core/tools 1.11
• Optimise MultiQC configuration for faster run-time on huge sample numbers
• Add information about SILVA licensing when removing rRNA to usage.md
• Fixed ansi colours for pipeline summary, added summary logs of alignment results
• [#281] - Add nag to cite the pipeline in summary
• [#302] - Fixed MDS plot axis labels
• [#338] - Add option for turning on/off STAR command line option (--sjdbGTFfile)
• [#344] - Added multi-core TrimGalore support
• [#351] - Fixes missing Qualimap parameter -p
• [#353] - Fixes an issue where MultiQC fails to run with --skip_biotype_qc option
• [#357] - Fixes broken links
• [#362] - Fix error with gzipped annotation file
• [#384] - Changed SortMeRNA reference dbs path to use stable URLs (v4.2.0)
• [#396] - Deterministic mapping for STAR aligner
• [#412] - Fix Qualimap not being passed on correct strand-specificity parameter
• [#413] - Fix STAR unmapped reads not output
• [#434] - Fix typo reported for work-dir
• [#437] - FastQC uses correct number of threads now
• [#440] - Fixed issue where featureCounts process fails when setting --fc_count_type to gene
• [#452] - Fix --gff input bug
• [#345] - Fixes label name in FastQC process
• [#391] - Make publishDir mode configurable
• [#431] - Update AWS GitHub actions workflow with organization level secrets
• [#435] - Fix a bug where gzipped references were not extracted when --additional_fasta was not specified
• [#435] - Fix a bug where merging of RSEM output would fail if only one fastq provided as input
• [#435] - Correct RSEM output name (was saving counts but calling them TPMs; now saving both properly labelled)
• [#436] - Fix a bug where the RSEM reference could not be built
• [#458] - Fix TMP_DIR for process MarkDuplicates and Qualimap

Parameters

Updated

Old parameter	New parameter
--reads	--input
--igenomesIgnore	--igenomes_ignore
--removeRiboRNA	--remove_ribo_rna
--rRNA_database_manifest	--ribo_database_manifest
--save_nonrRNA_reads	--save_non_ribo_reads
--saveAlignedIntermediates	--save_align_intermeds
--saveReference	--save_reference
--saveTrimmed	--save_trimmed
--saveUnaligned	--save_unaligned
--skipAlignment	--skip_alignment
--skipBiotypeQC	--skip_biotype_qc
--skipDupRadar	--skip_dupradar
--skipFastQC	--skip_fastqc
--skipMultiQC	--skip_multiqc
--skipPreseq	--skip_preseq
--skipQC	--skip_qc
--skipQualimap	--skip_qualimap
--skipRseQC	--skip_rseqc
--skipTrimming	--skip_trimming
--stringTieIgnoreGTF	--stringtie_ignore_gtf

Added

• --additional_fasta - FASTA file to concatenate to genome FASTA file e.g. containing spike-in sequences
• --deseq2_vst - Use vst transformation instead of rlog with DESeq2
• --enable_conda - Run this workflow with Conda. You can also use '-profile conda' instead of providing this parameter
• --min_mapped_reads - Minimum percentage of uniquely mapped reads below which samples are removed from further processing
• --multiqc_title - MultiQC report title. Printed as page header, used for filename if not otherwise specified
• --public_data_ids - File containing SRA/ENA/GEO identifiers one per line in order to download their associated FastQ files
• --publish_dir_mode - Method used to save pipeline results to output directory
• --rsem_index - Path to directory or tar.gz archive for pre-built RSEM index
• --rseqc_modules - Specify the RSeQC modules to run
• --save_merged_fastq - Save FastQ files after merging re-sequenced libraries in the results directory
• --save_umi_intermeds - If this option is specified, intermediate FastQ and BAM files produced by UMI-tools are also saved in the results directory
• --skip_bigwig - Skip bigWig file creation
• --skip_deseq2_qc - Skip DESeq2 PCA and heatmap plotting
• --skip_featurecounts - Skip featureCounts
• --skip_markduplicates - Skip picard MarkDuplicates step
• --skip_sra_fastq_download - Only download metadata for public data database ids and don't download the FastQ files
• --skip_stringtie - Skip StringTie
• --star_ignore_sjdbgtf - See #338
• --umitools_bc_pattern - The UMI barcode pattern to use e.g. 'NNNNNN' indicates that the first 6 nucleotides of the read are from the UMI
• --umitools_extract_method - UMI pattern to use. Can be either 'string' (default) or 'regex'
• --with_umi - Enable UMI-based read deduplication

Removed

• --awsqueue can now be provided via nf-core/configs if using AWS
• --awsregion can now be provided via nf-core/configs if using AWS
• --compressedReference now auto-detected
• --markdup_java_options in favour of updating centrally on nf-core/modules
• --project parameter from old NGI template
• --readPaths is not required since these are provided from the input samplesheet
• --sampleLevel not required
• --singleEnd is now auto-detected from the input samplesheet
• --skipEdgeR qc not performed by DESeq2 instead
• --star_memory in favour of updating centrally on nf-core/modules if required
• Strandedness is now specified at the sample-level via the input samplesheet
  • --forwardStranded
  • --reverseStranded
  • --unStranded
  • --pico

Software dependencies

Note, since the pipeline is now using Nextflow DSL2, each process will be run with its own Biocontainer. This means that on occasion it is entirely possible for the pipeline to be using different versions of the same tool. However, the overall software dependency changes compared to the last release have been listed below for reference.

Dependency	Old version	New version
bioconductor-dupradar	1.14.0	1.18.0
bioconductor-summarizedexperiment	1.14.0	1.18.1
bioconductor-tximeta	1.2.2	1.6.3
fastqc	0.11.8	0.11.9
gffread	0.11.4	0.12.1
hisat2	2.1.0	2.2.0
multiqc	1.7	1.9
picard	2.21.1	2.23.8
qualimap	2.2.2c	2.2.2d
r-base	3.6.1	4.0.3
salmon	0.14.2	1.3.0
samtools	1.9	1.10
sortmerna	2.1b	4.2.0
stringtie	2.0	2.1.4
subread	1.6.4	2.0.1
trim-galore	0.6.4	0.6.6
bedtools	-	2.29.2
bioconductor-biocparallel	-	1.22.0
bioconductor-complexheatmap	-	2.4.2
bioconductor-deseq2	-	1.28.0
bioconductor-tximport	-	1.16.0
perl	-	5.26.2
python	-	3.8.3
r-ggplot2	-	3.3.2
r-optparse	-	1.6.6
r-pheatmap	-	1.0.12
r-rcolorbrewer	-	1.1_2
rsem	-	1.3.3
ucsc-bedgraphtobigwig	-	377
umi_tools	-	1.0.1
bioconductor-edger	-	-
deeptools	-	-
matplotlib	-	-
r-data.table	-	-
r-gplots	-	-
r-markdown	-	-

NB: Dependency has been updated if both old and new version information is present.
NB: Dependency has been added if just the new version information is present.
NB: Dependency has been removed if version information isn't present.