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sdparekh / zUMIs

zUMIs: A fast and flexible pipeline to process RNA sequencing data with UMIs
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EXITING because of fatal PARAMETERS error: sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile #365

Open hxyyxh opened 1 year ago

hxyyxh commented 1 year ago

Hello, I am trying to use the example dataset to test zUMI. I cannot open the index provided, so I set up the index myself, but I have encountered some errors. Could you please give me some suggestions? Thank you very much!

/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/zUMIs-main/zUMIs.sh -y /cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b+gtf.yaml

You provided these parameters: YAML file: /cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b+gtf.yaml zUMIs directory: /cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/zUMIs-main STAR executable STAR samtools executable samtools pigz executable pigz Rscript executable Rscript RAM limit: 100 zUMIs version 2.9.7e

Tue Jul 11 14:52:08 CST 2023 WARNING: The STAR version used for mapping is 2.5.4b and the STAR index was created using the version 20201. This may lead to an error while mapping. If you encounter any errors at the mapping stage, please make sure to create the STAR index using STAR 2.5.4b. Filtering... Tue Jul 11 14:52:18 CST 2023 [1] "44 barcodes detected." [1] "203543 reads were assigned to barcodes that do not correspond to intact cells." Warning message: Using size aesthetic for lines was deprecated in ggplot2 3.4.0. Please use linewidth instead. [1] "Found 3 daughter barcodes that can be binned into 3 parent barcodes." [1] "Binned barcodes correspond to 389 reads." Mapping... [1] "2023-07-11 14:52:22 CST" [E::hts_open_format] Failed to open file "NA" : No such file or directory samtools view: failed to open "NA" for reading: No such file or directory Warning message: In data.table::fread(cmd = paste(samtools, "view", filtered_bams[1], : File '/tmp/RtmpHavZ27/file4f4a133311cc3' has size 0. Returning a NULL data.table. Usage: samtools cat [options] [... ] samtools cat [options] [... ]

Concatenate BAM or CRAM files, first those in , then those on the command line.

Options: -b FILE list of input BAM/CRAM file names, one per line -h FILE copy the header from FILE [default is 1st input file] -o FILE output BAM/CRAM --no-PG do not add a PG line --output-fmt FORMAT[,OPT[=VAL]]... Specify output format (SAM, BAM, CRAM) --output-fmt-option OPT[=VAL] Specify a single output file format option in the form of OPTION or OPTION=VALUE -@, --threads INT Number of additional threads to use [0] --verbosity INT Set level of verbosity [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory

EXITING because of fatal PARAMETERS error: sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile SOLUTION: specify sjdbOverhang>0, ideally readmateLength-1 Jul 11 14:52:24 ...... FATAL ERROR, exiting [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory

EXITING because of fatal PARAMETERS error: sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile SOLUTION: specify sjdbOverhang>0, ideally readmateLength-1 Jul 11 14:52:24 ...... FATAL ERROR, exiting [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory [E::hts_open_format] Failed to open file "NULL" : No such file or directory samtools view: failed to open "NULL" for reading: No such file or directory

EXITING because of fatal PARAMETERS error: sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile SOLUTION: specify sjdbOverhang>0, ideally readmateLength-1 Jul 11 14:52:24 ...... FATAL ERROR, exiting cp: cannot stat ‘/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/zUMIs_output/.tmpMap//tmp.2.5.4b_star_hg19+gtf.*.SJ.out.tab’: No such file or directory Usage: samtools cat [options] [... ] samtools cat [options] [... ]

Concatenate BAM or CRAM files, first those in , then those on the command line.

Options: -b FILE list of input BAM/CRAM file names, one per line -h FILE copy the header from FILE [default is 1st input file] -o FILE output BAM/CRAM --no-PG do not add a PG line --Usage: samtools cat [options] [... ] output-fmt FORMAT[,OPT[=VAL]]... Specify output format (SAM, BAM, CRAM) --output-fmt-option OPT[=VAL] Specify a single output file format option in the form of OPTION or OPTION=VALUE samtools cat [options] [... ]

-@, --threads INT Number of additional threads to use [0] --verbosity INT Set level of verbosity Concatenate BAM or CRAM files, first those in , then those on the command line.

Options: -b FILE list of input BAM/CRAM file names, one per line -h FILE copy the header from FILE [default is 1st input file] -o FILE output BAM/CRAM --no-PG do not add a PG line --output-fmt FORMAT[,OPT[=VAL]]... Specify output format (SAM, BAM, CRAM) --output-fmt-option OPT[=VAL] Specify a single output file format option in the form of OPTION or OPTION=VALUE -@, --threads INT Number of additional threads to use [0] --verbosity INT Set level of verbosity Tue Jul 11 14:52:24 CST 2023 Counting... [1] "2023-07-11 14:52:36 CST" [1] "1.5e+08 Reads per chunk" [1] "Loading reference annotation from:" [1] "/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b_star_hg19+gtf.final_annot.gtf" [E::hts_open_format] Failed to open file "/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b_star_hg19+gtf.filtered.tagged.Aligned.out.bam" : No such file or directory samtools view: failed to open "/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b_star_hg19+gtf.filtered.tagged.Aligned.out.bam" for reading: No such file or directory [E::hts_open_format] Failed to open file "/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b_star_hg19+gtf.filtered.tagged.Aligned.out.bam" : No such file or directory samtools view: failed to open "/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/2.5.4b_star_hg19+gtf.filtered.tagged.Aligned.out.bam" for reading: No such file or directory Error in h(simpleError(msg, call)) : error in evaluating the argument 'x' in selecting a method for function 'gsub': object 'chr' not found Calls: .makeSAF ... [.data.table -> eval -> eval -> gsub -> .handleSimpleError -> h In addition: Warning message: In data.table::fread(bread, col.names = c("chr", "len"), header = F) : File '/tmp/Rtmpn3XvE5/file4f4e3717e90a7' has size 0. Returning a NULL data.table. Execution halted Tue Jul 11 14:52:58 CST 2023 Loading required package: yaml Loading required package: Matrix [1] "loomR found" Error in gzfile(file, "rb") : cannot open the connection Calls: rds_to_loom -> readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/zUMIs_output/expression/2.5.4b_star_hg19+gtf.dgecounts.rds', probable reason 'No such file or directory' Execution halted Tue Jul 11 14:53:01 CST 2023 Descriptive statistics... [1] "I am loading useful packages for plotting..." [1] "2023-07-11 14:53:01 CST" Error in gzfile(file, "rb") : cannot open the connection Calls: readRDS -> gzfile In addition: Warning message: In gzfile(file, "rb") : cannot open compressed file '/cluster/groups/Chris-Lab/common/Xinyang/Data/SS3-p/zumi/test/2.5.4b_star_hg19/+gtf/zUMIs_output/expression/2.5.4b_star_hg19+gtf.dgecounts.rds', probable reason 'No such file or directory' Execution halted Tue Jul 11 14:53:07 CST 2023

hxyyxh commented 1 year ago

I used UCSC hg19 to build index, STAR version is 2.5.4b, yaml file is here.

2.5.4b+gtf.zip