Auto Chemistry Detection Failing and FIFO File Generation Problem

[Sandman-1]

Hello! Thank you for such an excellent software.

I am trying to run CeleScope v2.0.7 on raw fastqs from GSE203360, but it throws me the following error.

• Exception: Auto chemistry detection failed! --chemistry auto can auto-detect scopeV2 mRNA, scopeV3 mRNA, full length VDJ mRNA(flv_rna) and full length VDJ(flv). You need to explicitly use --chemistry scopeV1 for legacy chemistry scopeV1. --chemistry customized is used for user defined combinations that you need to provide --pattern, --whitelist and --linker at the same time.

I don't know what the kit version is for GSE203360, but I tried supplying the latest version (scopeV3.0.1). It then gave me the following error.

• Exiting because of FATAL ERROR: could not create FIFO file .//Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53/01.starsolo/Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53__STARtmp/tmp.fifo.read1 SOLUTION: check the if run directory supports FIFO files. If run partition does not support FIFO (e.g. Windows partitions FAT, NTFS), re-run on a Linux partition, or point --outTmpDir to a Linux partition.

Could you help me navigate these errors? I am working with WSL2 on my Windows computer.

[zhouyiqi91]

1. I have checked one run(SRR19312208) of GSE203360 and it is scopeV1.
2. Exiting because of FATAL ERROR: could not create FIFO file Please take a look at https://github.com/alexdobin/STAR/issues/1776 You can add extra STAR params by adding "--STAR_param --outTmpDir some_dir " to multi_rna

[Sandman-1]

Thank you! I am getting this error after adding the parameter, however. I add it to the end of the starsolo command within each sample-specific shell file.

• celescope rna starsolo: error: argument --STAR_param: expected one argument

[zhouyiqi91]

Sorry. It needs to be double quoted, like this --STAR_param "--outTmpDir some_dir"

[Sandman-1]

You are a lifesaver. Thank you so much! Curious, how do you know which GexScope kit version a run is?

[zhouyiqi91]

By checking the pattern of R1 reads. https://trace.ncbi.nlm.nih.gov/Traces/?view=run_browser&page_size=10&acc=SRR19312208&display=reads https://github.com/singleron-RD/CeleScope/blob/master/doc/chemistry.md

[Sandman-1]

Hello! I tried running the pipeline overnight, and it gave me this error when I woke up.

• /usr/bin/STAR: line 7: 149706 Segmentation fault "${cmd}" "$@" Traceback (most recent call last): File "/home/hebbale/.local/bin/celescope", line 8, in sys.exit(main()) File "/home/hebbale/.local/lib/python3.10/site-packages/celescope/celescope.py", line 54, in main args.func(args) File "/home/hebbale/.local/lib/python3.10/site-packages/celescope/tools/starsolo.py", line 186, in starsolo q30_cb, q30_umi, chemistry = runner.run() File "/home/hebbale/.local/lib/python3.10/site-packages/celescope/tools/starsolo.py", line 177, in run self.run_starsolo() File "/home/hebbale/.local/lib/python3.10/site-packages/celescope/tools/starsolo.py", line 133, in run_starsolo subprocess.check_call(cmd, shell=True) File "/usr/lib/python3.10/subprocess.py", line 369, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command 'STAR \ --genomeDir /mnt/c/Users/hebbale/Downloads/human_GRCh38_optimized_v2_celescope \ --readFilesIn Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53/Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53_R2.fq.gz Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53/Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53_R1.fq.gz \ --readFilesCommand zcat \ --soloCBwhitelist None \ --soloCellFilter EmptyDrops_CR 3000 0.99 10 45000 90000 500 0.01 20000 0.001 10000 \ --outFileNamePrefix .//Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female_53/01.starsolo/Lung_GSE203360_IAC_mGGO_P1_Ground_Glass_Female53 \ --runThreadN 8 \ --clip3pAdapterSeq AAAAAAAAAAAA \ --outFilterMatchNmin 50 \ --soloFeatures Gene GeneFull_Ex50pAS \ --outSAMattributes NH HI nM AS CR UR CB UB GX GN \ --soloType CB_UMI_Simple \ --soloCBstart 1 --soloCBlen 12 \ --soloUMIstart 13 --soloUMIlen 8 \ --soloCBmatchWLtype 1MM \ --outSAMtype BAM SortedByCoordinate \ --soloCellReadStats Standard \ --soloBarcodeReadLength 0 \ --outTmpDir /home/hebbale/tmp' returned non-zero exit status 139.

I don't understand what the segmentation fault refers to, or if it's a problem with the temporary directory I assigned file construction to in my linux partition, or if I am missing something else entirely. However, I think this should be the last error in the pipeline to run celescope properly. Could you please help me?

[zhouyiqi91]

Found an exit code 139 issue in STAR issue. https://github.com/alexdobin/STAR/issues/887 But it shouldn't be the same reason here. I see that you are not using a conda environment; I'm not sure what version of STAR you are using. Could this be causing the problem?