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README.md

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@@ -32,28 +32,29 @@ You can find numerous talks on the [nf-core events page](https://nf-co.re/events
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> The SRA download functionality has been removed from the pipeline (`>=3.2`) and ported to an independent workflow called [nf-core/fetchngs](https://nf-co.re/fetchngs). You can provide `--nf_core_pipeline rnaseq` when running nf-core/fetchngs to download and auto-create a samplesheet containing publicly available samples that can be accepted directly as input by this pipeline.
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1. Merge re-sequenced FastQ files ([`cat`](http://www.linfo.org/cat.html))
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2. Read QC ([`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/))
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3. UMI extraction ([`UMI-tools`](https://github.com/CGATOxford/UMI-tools))
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4. Adapter and quality trimming ([`Trim Galore!`](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/))
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5. Removal of genome contaminants ([`BBSplit`](http://seqanswers.com/forums/showthread.php?t=41288))
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6. Removal of ribosomal RNA ([`SortMeRNA`](https://github.com/biocore/sortmerna))
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7. Choice of multiple alignment and quantification routes:
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2. Sub-sample FastQ files and auto-infer strandedness ([`fq`](https://github.com/stjude-rust-labs/fq), [`Salmon`](https://combine-lab.github.io/salmon/))
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3. Read QC ([`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/))
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4. UMI extraction ([`UMI-tools`](https://github.com/CGATOxford/UMI-tools))
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5. Adapter and quality trimming ([`Trim Galore!`](https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/))
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6. Removal of genome contaminants ([`BBSplit`](http://seqanswers.com/forums/showthread.php?t=41288))
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7. Removal of ribosomal RNA ([`SortMeRNA`](https://github.com/biocore/sortmerna))
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9. Choice of multiple alignment and quantification routes:
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1. [`STAR`](https://github.com/alexdobin/STAR) -> [`Salmon`](https://combine-lab.github.io/salmon/)
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2. [`STAR`](https://github.com/alexdobin/STAR) -> [`RSEM`](https://github.com/deweylab/RSEM)
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3. [`HiSAT2`](https://ccb.jhu.edu/software/hisat2/index.shtml) -> **NO QUANTIFICATION**
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8. Sort and index alignments ([`SAMtools`](https://sourceforge.net/projects/samtools/files/samtools/))
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9. UMI-based deduplication ([`UMI-tools`](https://github.com/CGATOxford/UMI-tools))
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10. Duplicate read marking ([`picard MarkDuplicates`](https://broadinstitute.github.io/picard/))
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11. Transcript assembly and quantification ([`StringTie`](https://ccb.jhu.edu/software/stringtie/))
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12. Create bigWig coverage files ([`BEDTools`](https://github.com/arq5x/bedtools2/), [`bedGraphToBigWig`](http://hgdownload.soe.ucsc.edu/admin/exe/))
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13. Extensive quality control:
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9. Sort and index alignments ([`SAMtools`](https://sourceforge.net/projects/samtools/files/samtools/))
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10. UMI-based deduplication ([`UMI-tools`](https://github.com/CGATOxford/UMI-tools))
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11. Duplicate read marking ([`picard MarkDuplicates`](https://broadinstitute.github.io/picard/))
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12. Transcript assembly and quantification ([`StringTie`](https://ccb.jhu.edu/software/stringtie/))
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13. Create bigWig coverage files ([`BEDTools`](https://github.com/arq5x/bedtools2/), [`bedGraphToBigWig`](http://hgdownload.soe.ucsc.edu/admin/exe/))
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14. Extensive quality control:
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1. [`RSeQC`](http://rseqc.sourceforge.net/)
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2. [`Qualimap`](http://qualimap.bioinfo.cipf.es/)
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3. [`dupRadar`](https://bioconductor.org/packages/release/bioc/html/dupRadar.html)
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4. [`Preseq`](http://smithlabresearch.org/software/preseq/)
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5. [`DESeq2`](https://bioconductor.org/packages/release/bioc/html/DESeq2.html)
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14. Pseudo-alignment and quantification ([`Salmon`](https://combine-lab.github.io/salmon/); _optional_)
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15. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks ([`MultiQC`](http://multiqc.info/), [`R`](https://www.r-project.org/))
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15. Pseudo-alignment and quantification ([`Salmon`](https://combine-lab.github.io/salmon/); _optional_)
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16. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks ([`MultiQC`](http://multiqc.info/), [`R`](https://www.r-project.org/))
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> **Warning**
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> Quantification isn't performed if using `--aligner hisat2` due to the lack of an appropriate option to calculate accurate expression estimates from HISAT2 derived genomic alignments. However, you can use this route if you have a preference for the alignment, QC and other types of downstream analysis compatible with the output of HISAT2.
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The pipeline workflow diagram was designed by Sarah Guinchard ([@G-Sarah](https://github.com/G-Sarah)) and James Fellows Yates ([@jfy133](https://github.com/jfy133)).
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Many thanks to other who have helped out along the way too, including (but not limited to):
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[@MatthiasZepper](https://github.com/MatthiasZepper),
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[@maxulysse](https://github.com/maxulysse),
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[@robsyme](https://github.com/robsyme),
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[@Galithil](https://github.com/Galithil),
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[@pditommaso](https://github.com/pditommaso),
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[@orzechoj](https://github.com/orzechoj),

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