# salmonKallistoMtxTo10x not working **URL:** \ **Category:** Uncategorized\ **Tags:** workflow, salmon\ **Created:** [February 1, 2022, 7:04pm UTC](https://help.galaxyproject.org/t/salmonkallistomtxto10x-not-working/7437 "2022-02-01T19:04:26Z")\ **Posts on this page:** 4\ **Page:** 1
**Author:** ![pa.saunders](https://avatars.discourse-cdn.com/v4/letter/p/3ec8ea/32.png) [@pa.saunders](https://help.galaxyproject.org/u/pa.saunders)\ **Post date:** [February 1, 2022, 7:04pm UTC](https://help.galaxyproject.org/t/salmonkallistomtxto10x-not-working/7437/1 "2022-02-01T19:04:26Z")
I have been trying to perform a scRNA sequence experiment with the Galaxy tutorial [Generating a single cell matrix using Alevin](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/droplet-quantification-preprocessing/tutorial.html) and keep hanging up when I get to the SalmonKallistoMtxTo10x step. The failure happens both with the data set in the tutorial and with a smaller data set directly from 10x genomics. Details on data set and run parameters below. Any suggestion on how to get past this blockage would be helpful. The error output files are empty. > **[Galaxy Training: Generating a single cell matrix using Alevin](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/droplet-quantification-preprocessing/tutorial.html)** > > Training material for all kinds of transcriptomics analysis. 1k PBMCs from a Healthy Donor (v3 chemistry) Single Cell Gene Expression Dataset by Cell Ranger 3.0.0 Peripheral blood mononuclear cells (PBMCs) from a healthy donor (the same cells were used to generate pbmc\_1k\_v2, pbmc\_10k\_v3). PBMCs are primary cells with relatively small amounts of RNA (~1pg RNA/cell). • 1,222 cells detected • Sequenced on Illumina NovaSeq with approximately 54,000 reads per cell • 28bp read1 (16bp Chromium barcode and 12bp UMI), 91bp read2 (transcript), and 8bp I7 sample barcode • run with --expect-cells=1000 * * * GTF2GeneList extracts a complete annotation table or subsets thereof from an Ensembl GTF using rtracklayer (Galaxy Version 1.42.1+galaxy6) Ensembl GTF file Feature type for which to derive annotation transcript Field to place first in output table transcript\_id Suppress header line in output? Yes transcript\_id,gene\_id Append version to transcript identifiers? Yes Flag mitochondrial features? No Filter a FASTA-format cDNA file to match annotations? Yes Annotation field to match with sequences. transcript\_id Rename galaxy-pencil the annotation table to Map Rename galaxy-pencil the uncompressed filtered FASTA file to Filtered FASTA * * * Alevin Quantification and analysis of 3’ tagged-end single-cell sequencing data (Galaxy Version 1.3.0+galaxy2) Tool Parameters Input Parameter Value Select a reference transcriptome from your history or use a built-in index? history Transcripts fasta file 10 Filtered FASTA uncompressed (Hidden) Kmer length 31 Perfect Hash False Single or paired-end reads? paired 4 pbmc\_1k\_v3\_S1\_L001\_R1\_001.fastq.gz 5 pbmc\_1k\_v3\_S1\_L001\_R2\_001.fastq.gz Relative orientation of reads within a pair Mates are oriented toward each other (I = inward) Specify the strandedness of the reads read comes from the reverse strand (SR) protocol 10x chromium v3 Single Cell protocol Transcript to gene map file 9 Map Retrieve all output files True optional Whitelist file noDedup False dumpBfh False dumpFeatures True dumpUmiGraph False dumpMtx True forceCells Not available. expectCells Not available. numCellBootstraps Not available. minScoreFraction Not available. keepCBFraction 1.0 lowRegionMinNumBarcodes Not available. maxNumBarcodes Not available. freqThreshold 3 * * * SalmonKallistoMtxTo10x Transforms .mtx matrix and associated labels into a format compatible with tools expecting old-style 10X data (Galaxy Version 0.0.1+galaxy5) Tool Parameters Input Parameter Value .mtx-format matrix 11 quants\_mat.mtx Tab-delimited genes file 13 quants\_mat\_cols.txt Tab-delimited barcodes file 14 quants\_mat\_rows.txt Prefix to prepend to cell names / barcodes Empty ---
**Author:** ![Wendi\_B](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wendi_b/32/1193_2.png) [@Wendi\_B](https://help.galaxyproject.org/u/Wendi_B)\ **Post date:** [February 3, 2022, 1:33pm UTC](https://help.galaxyproject.org/t/salmonkallistomtxto10x-not-working/7437/2 "2022-02-03T13:33:35Z")
Hello! Yes absolutely - can you please share your history? ---
**Author:** ![rituu-vermaa](https://avatars.discourse-cdn.com/v4/letter/r/e47774/32.png) [@rituu-vermaa](https://help.galaxyproject.org/u/rituu-vermaa)\ **Post date:** [May 8, 2022, 5:51pm UTC](https://help.galaxyproject.org/t/salmonkallistomtxto10x-not-working/7437/3 "2022-05-08T17:51:31Z")
Hey @Wendy\_B I’ve been experiencing the same problem (details of the error below). As for the data, I used the one here: (Given on [Generating a single cell matrix using Alevin](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/droplet-quantification-preprocessing/tutorial.html) ) [https://zenodo.org/record/4574153/files/Experimental\_Design.tabular](https://zenodo.org/record/4574153/files/Experimental_Design.tabular) [https://zenodo.org/record/4574153/files/Mus\_musculus.GRCm38.100.gtf.gff](https://zenodo.org/record/4574153/files/Mus_musculus.GRCm38.100.gtf.gff) [https://zenodo.org/record/4574153/files/Mus\_musculus.GRCm38.cdna.all.fa.fasta](https://zenodo.org/record/4574153/files/Mus_musculus.GRCm38.cdna.all.fa.fasta) [https://zenodo.org/record/4574153/files/SLX-7632.TAAGGCGA.N701.s\_1.r\_1.fq-400k.fastq](https://zenodo.org/record/4574153/files/SLX-7632.TAAGGCGA.N701.s_1.r_1.fq-400k.fastq) [https://zenodo.org/record/4574153/files/SLX-7632.TAAGGCGA.N701.s\_1.r\_2.fq-400k.fastq](https://zenodo.org/record/4574153/files/SLX-7632.TAAGGCGA.N701.s_1.r_2.fq-400k.fastq) Can you suggest why there’s an error here? DETAILS OF ERROR: Execution resulted in the following messages: Fatal error: Exit code 1 () Tool generated the following standard error: Traceback (most recent call last): File “/cvmfs/main.galaxyproject.org/shed\_tools/toolshed.g2.bx.psu.edu/repos/ebi-gxa/salmon\_kallisto\_mtx\_to\_10x/e42c217a450f/salmon\_kallisto\_mtx\_to\_10x/salmonKallistoMtxTo10x.py”, line 71, in mmwrite(’%s/matrix.mtx’ % mtx\_out, umi\_counts.transpose()) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 101, in mmwrite MMFile().write(target, a, comment, field, precision, symmetry) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 451, in write stream, close\_it = self.\_open(target, ‘wb’) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 323, in \_open stream = open(filespec, mode) OSError: [Errno 30] Read-only file system: ‘.[//matrix.mtx](https://matrix.mtx)’ Galaxy job runner generated the following standard error: Traceback (most recent call last): File “/cvmfs/main.galaxyproject.org/shed\_tools/toolshed.g2.bx.psu.edu/repos/ebi-gxa/salmon\_kallisto\_mtx\_to\_10x/e42c217a450f/salmon\_kallisto\_mtx\_to\_10x/salmonKallistoMtxTo10x.py”, line 71, in mmwrite(’%s/matrix.mtx’ % mtx\_out, umi\_counts.transpose()) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 101, in mmwrite MMFile().write(target, a, comment, field, precision, symmetry) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 451, in write stream, close\_it = self.\_open(target, ‘wb’) File “/usr/local/lib/python3.8/site-packages/scipy/io/mmio.py”, line 323, in \_open stream = open(filespec, mode) OSError: [Errno 30] Read-only file system: ‘.[//matrix.mtx](https://matrix.mtx)’ ---
**Author:** ![jennaj](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/jennaj/32/27_2.png) [@jennaj](https://help.galaxyproject.org/u/jennaj)\ **Post date:** [May 11, 2022, 10:22pm UTC](https://help.galaxyproject.org/t/salmonkallistomtxto10x-not-working/7437/4 "2022-05-11T22:22:24Z")
Hi @pa.saunders and @rituu-vermaa > [@rituu-vermaa](#): > > OSError: [Errno 30] Read-only file system: ‘.[//matrix.mtx](https://matrix.mtx/)’ This tool `SalmonKallistoMtxTo10x` needs some tool developer then administrative changes to work at [UseGalaxy.org](http://UseGalaxy.org). I suspect `Alevin` has this problem as well (not confirmed yet). Please try running this tutorial, or any workflows based on it, at the [UseGalaxy.eu](http://UseGalaxy.eu) server instead for now. If you need to move existing data between the servers, the how-to is in this [FAQ](https://training.galaxyproject.org/training-material/faqs/galaxy/#moving-datasets-between-galaxy-servers). Ps: @rituu-vermaa , Thanks for submitting the bug reports. I replied to some of those already re: mixed up inputs. That is a bit common with this tutorial since it repeats groups of steps with slightly different criteria a few times, which is arguably confusing but unavoidable when doing the work step-by-step and not with a workflow/collection yet. You could consider adding in [dataset #tags](https://training.galaxyproject.org/training-material/faqs/galaxy/datasets_add_tag.html) to help keep track of the different runs ([example screenshot in that tutorial with tags included](https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/droplet-quantification-preprocessing/tutorial.html#adding-batch-metadata)). Later on when using a workflow and collections, tags or not, that will be less likely to happen, and is one reason why both of those functions are popular and worth learning about (search the GTN tutorials with keywords for help with those: “collection” and/or “workflow”). Hope that helps, and apologies for the confusing tool trouble on top of a complicated tutorial!