nextflow run nf-core/riboseq -profile docker --input Sample_sheet.csv --outdir Riboseq_out --genome mm10 --trimmer fastp

ERROR ~ Error executing process > 'NFCORE_RIBOSEQRIBOSEQFASTQ_ALIGN_STAR:STAR_ALIGN (Treated)' Caused by: Process

NFCORE_RIBOSEQ:RIBOSEQ:FASTQ_ALIGN_STAR:STAR_ALIGN (Treated)

terminated with an error exit status (105) Command executed: STAR \ --genomeDir star \ --readFilesIn input1/Treated_1.non_rRNA.fastq.gz input2/Treated_2.non_rRNA.fastq.gz \ --runThreadN 12 \ --outFileNamePrefix Treated. \ \ --sjdbGTFfile genome.filtered.gtf \ --outSAMattrRGline 'ID:Treated' 'SM:Treated' \ --alignSJDBoverhangMin 1 --alignEndsType EndToEnd --outFilterMultimapNmax 20 --outSAMattributes All --outSAMstrandField intronMotif --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --quantTranscriptomeBan Singleend --readFilesCommand zcat --runRNGseed 0 --twopassMode Basic if [ -f Treated.Unmapped.out.mate1 ]; then mv Treated.Unmapped.out.mate1 Treated.unmapped_1.fastq gzip Treated.unmapped_1.fastq fi if [ -f Treated.Unmapped.out.mate2 ]; then mv Treated.Unmapped.out.mate2 Treated.unmapped_2.fastq gzip Treated.unmapped_2.fastq fi cat <-END_VERSIONS versions.yml "NFCORE_RIBOSEQRIBOSEQFASTQ_ALIGN_STAR:STAR_ALIGN": star: $(STAR --version | sed -e "s/STAR_//g") samtools: $(echo $(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*$//') gawk: $(echo $(gawk --version 2>&1) | sed 's/^.*GNU Awk //; s/, .*$//') END_VERSIONS Command exit status: 105 Command output: STAR --genomeDir star --readFilesIn input1/Treated_1.non_rRNA.fastq.gz input2/Treated_2.non_rRNA.fastq.gz --runThreadN 12 --outFileNamePrefix Treated. --sjdbGTFfile genome.filtered.gtf --outSAMattrRGline ID:Treated SM:Treated --alignSJDBoverhangMin 1 --alignEndsType EndToEnd --outFilterMultimapNmax 20 --outSAMattributes All --outSAMstrandField intronMotif --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --quantTranscriptomeBan Singleend --readFilesCommand zcat --runRNGseed 0 --twopassMode Basic STAR version: 2.7.10a compiled: 2022-01-14T185000-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 15 023416 ..... started STAR run Mar 15 023416 ..... loading genome Command error: STAR --genomeDir star --readFilesIn input1/Treated_1.non_rRNA.fastq.gz input2/Treated_2.non_rRNA.fastq.gz --runThreadN 12 --outFileNamePrefix Treated. --sjdbGTFfile genome.filtered.gtf --outSAMattrRGline ID:Treated SM:Treated --alignSJDBoverhangMin 1 --alignEndsType EndToEnd --outFilterMultimapNmax 20 --outSAMattributes All --outSAMstrandField intronMotif --outSAMtype BAM Unsorted --quantMode TranscriptomeSAM --quantTranscriptomeBan Singleend --readFilesCommand zcat --runRNGseed 0 --twopassMode Basic STAR version: 2.7.10a compiled: 2022-01-14T185000-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source Mar 15 023416 ..... started STAR run Mar 15 023416 ..... loading genome EXITING because of FATAL ERROR: Genome version: 20201 is INCOMPATIBLE with running STAR version: 2.7.10a SOLUTION: please re-generate genome from scratch with running version of STAR, or with version: 2.7.4a Mar 15 023416 ...... FATAL ERROR, exiting Work dir: /media/manoj/Data/Manoj/Riboseq/work/cb/62ddc13bd0836e9601e1929b6abe68 Container: quay.io/biocontainers/mulled-v2-1fa26d1ce03c295fe2fdcf85831a92fbcbd7e8c2:ded3841da0194af2701c780e9b3d653a85d27489-0 Tip: when you have fixed the problem you can continue the execution adding the option

-resume

to the run command line -- Check '.nextflow.log' file for details is there any way to change the start index. because I'm facing an issue with it. enclosed my log file for more detailed error

hello, this looks like an incompatibility between the STAR version used for igenomes mm10 and the STAR version in the container i.e

2.7.10a

. For now you can just provide as input only the GTF and FASTA files, but not the STAR index, and the pipeline will generate a new STAR index during execution from your annotation files.

I did not Provide any input file. For star index

You provided the mm10 with the genome flag. That loads the star_genome pre-generated in igenomes. Check the documentation and the igenomes.config file provided with the code.

Thank you will look into it

Hi @Manojkumar K have you managed to sort this out? I have the same problem

no periodic read length found

Thanks

Hi @Miriam, typically this kind of error comes about due to sample mix ups where you try to run detection methods on RNA-Seq data rather than Ribo-Seq. The other option is that you may not have correctly trimmed your reads and the addition bases from the adapter/umi prevent periodic signal. The other possibility is that your data was produced with a protocol that uses a less aggressive RNase digestion using something like MNase. This won't produce data with high periodicity when using typical workflows but you could look into the methods produced by Michael VanInseberghe in his single cell ribo-seq work or the tool 'psite' https://www.biorxiv.org/content/10.1101/2023.06.27.546788v1

Hi @Jack Tierney Thank you for your quick response. I've been told that they have prepared RiboSeq with a kit, but I don't know which one, so it's unlikely that it's an RNASeq mix up. The error is generated by the pipeline, so the trimming performed is the default. I'll dig deeper with my collaborator thank you!

Have a look at the earlier reports like FastQC or TrimGalore!. It'll give you quick answers. If you need a hand interpretting them I can have a look and talk you through what I'd look at

@Jack Tierney I'd love to take up you offer of what to look on the QC reports. I can see that after trimming I get a bimodal sequence length distribution with a major peak around 34-25 bps and a minor peak at 28-29. There's no adapter content, but there are overrepresented sequences that map to yeast ribosomal rRNAs. I'm unsure what else to look at. Thanks

You may just have rRNA contamination then. You will see in the main channel that Jonathan and others are working on a new version with RiboDetector as an option. It may be worth waiting until that is integrated. Out of interest, how did sortMeRNA do on removing rRNA from your samples?

I'm unsure as I'm no familiar with the output.

Well thats the role that sortMeRNA does. We have been discussing the addition of RiboDetector and alternately bowtie2 as additional methods

It looks like the mapping was also quite low 29% with ~60% of reads unmapped.

Started job on |	Nov 28 12:20:10
                             Started mapping on |	Nov 28 12:30:49
                                    Finished on |	Nov 28 12:43:29
       Mapping speed, Million of reads per hour |	373.69

                          Number of input reads |	78889821
                      Average input read length |	27
                                    UNIQUE READS:
                   Uniquely mapped reads number |	23014529
                        Uniquely mapped reads % |	29.17%
                          Average mapped length |	27.82
                       Number of splices: Total |	5899998
            Number of splices: Annotated (sjdb) |	5621977
                       Number of splices: GT/AG |	5875647
                       Number of splices: GC/AG |	4469
                       Number of splices: AT/AC |	1348
               Number of splices: Non-canonical |	18534
                      Mismatch rate per base, % |	0.99%
                         Deletion rate per base |	0.00%
                        Deletion average length |	2.32
                        Insertion rate per base |	0.00%
                       Insertion average length |	1.11
                             MULTI-MAPPING READS:
        Number of reads mapped to multiple loci |	9358060
             % of reads mapped to multiple loci |	11.86%
        Number of reads mapped to too many loci |	21199
             % of reads mapped to too many loci |	0.03%
                                  UNMAPPED READS:
  Number of reads unmapped: too many mismatches |	78
       % of reads unmapped: too many mismatches |	0.00%
            Number of reads unmapped: too short |	7255559
                 % of reads unmapped: too short |	9.20%
                Number of reads unmapped: other |	39240396
                     % of reads unmapped: other |	49.74%
                                  CHIMERIC READS:
                       Number of chimeric reads |	0
                            % of chimeric reads |	0.00%

Based on that log it looks~46% of your reads are rRNA. But these were removed by sortmerna. So your remaining reads somehow still don’t satisfy periodicity - either because there’s still contaminants left, or the footprints themselves don’t look like they satisfy periodicity. Do you know if this library was produced with RNAse I?

what's strange, is that with an in-house script to align first to rRNA and then to the same yeast reference that I'm using with the pipeline, I do see certain periodicity.

Ira, based on the params we use for STAR the low mapping could be explained by UMI, barcodes, adapters still on the reads right?

Ira, I don't know, I can ask my collaborator. I know we don't have UMIs on the data but I'll ask about the use of RNAse I.

Miriam that looks pretty periodic to me. What is _fa?

Yepp if there’s any/ too many bases leftover from adaptors (or UMIs) or even non templated bases our end to end mapping and settings will hit them pretty hard. So I would definitely first check the reads are preprocessed correctly

formaldehyde treatment at low conc, my collaborator wanted to see if he could get rid off the biass that you see with chx around the ATG

I think in this case one of the easiest things would be to take the BAMs outputs from this pipeline , not your custom script ones, and see if you can replicate the periodicity around the AUG as in your plot. This will tell us if at least the successfully mapped reads are any good

My collaborator says that he is using RNAse I

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