EXITING because of fatal PARAMETERS error: sjdbOverhang <=0 while junctions are inserted on the fly with --sjdbFileChrStartEnd or/and --sjdbGTFfile

[hxyyxh]

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] <in1.bam> [... <inN.bam>]
samtools cat [options] <in1.cram> [... <inN.cram>]

Concatenate BAM or CRAM files, first those in <bamlist.fofn>, 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] <in1.bam> [... <inN.bam>]
samtools cat [options] <in1.cram> [... <inN.cram>]

Concatenate BAM or CRAM files, first those in <bamlist.fofn>, 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] <in1.bam> [... <inN.bam>]
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] <in1.cram> [... <inN.cram>]

-@, --threads INT
Number of additional threads to use [0]
--verbosity INT
Set level of verbosity
Concatenate BAM or CRAM files, first those in <bamlist.fofn>, 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]

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

2.5.4b+gtf.zip