cellranger 7.0.0 pipestance failed, when executing feature barcode

[sagrikachugh]

I am trying to run Gene expression with antibody capture and get the following error, Could anyone please help how to solve this? Please note I am using version 7.0.1 not 7.0.0 as stated in the title. Also, I have downloaded the mouse reference genome from cellranger website, I assume there is some issue with the gene index but I am not sure how to approach this error to solve it.

Here is my log:

Martian Runtime - v4.0.8 2023-09-15 14:53:48 [jobmngr] WARNING: User-supplied amount 100 GB is higher than the detected cgroup memory limit of 60.3 GB Serving UI at http://scgn08.svi.edu.au:38871?auth=RmZ4nvPW_DJvM0gaEIOWvMeFNQopulAublW_iPlMY3A

Running preflight checks (please wait)... Checking feature definition file... Checking sample info... Checking FASTQ folder... Checking reference... Checking reference_path (/mnt/beegfs/mccarthy/backed_up/general/schugh/Projects/Purton_lab/reference_genome/mm10-new/refdata-gex-mm10-2020-A) on scgn08.svi.edu.au... Checking optional arguments... mro: v4.0.8 mrp: v4.0.8 Anaconda: Python 3.8.12 numpy: 1.21.2 scipy: 1.7.3 pysam: 0.16.0.1

[error] Pipestance failed. Error log at: svi_sample1/SC_RNA_COUNTER_CS/CELLRANGER_PREFLIGHT/fork0/chnk0-u18b403e35f/_errors

Log message: Traceback (most recent call last): File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/python/cellranger/reference.py", line 655, in load_from_reference subprocess.check_output(cmd, stderr=subprocess.STDOUT) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/anaconda/lib/python3.8/subprocess.py", line 415, in check_output return run(*popenargs, stdout=PIPE, timeout=timeout, check=True, File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/anaconda/lib/python3.8/subprocess.py", line 516, in run raise CalledProcessError(retcode, process.args, subprocess.CalledProcessError: Command '[b'/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/bin/gtf_to_gene_index', b'/mnt/beegfs/mccarthy/backed_up/general/schugh/Projects/Purton_lab/reference_genome/mm10-new/refdata-gex-mm10-2020-A', b'gene_index.json']' returned non-zero exit status 101.

The above exception was the direct cause of the following exception:

Traceback (most recent call last): File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/martian/adapters/python/martian_shell.py", line 680, in _main stage.main() File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/martian/adapters/python/martian_shell.py", line 639, in main self._run(lambda: self._module.main(args, outs)) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/martian/adapters/python/martian_shell.py", line 596, in _run cmd() File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/external/martian/adapters/python/martian_shell.py", line 639, in self._run(lambda: self._module.main(args, outs)) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/mro/rna/stages/common/cellranger_preflight/init.py", line 60, in main run_preflight_checks(args) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/mro/rna/stages/common/cellranger_preflight/init.py", line 34, in run_preflight_checks cr_preflight.check_sample_info( File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/python/cellranger/preflight.py", line 633, in check_sample_info feature_ref = try_load_feature_ref(reference_path, feature_ref_path) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/python/cellranger/preflight.py", line 312, in try_load_feature_ref feature_ref = rna_feature_ref.from_transcriptome_and_csv( File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/python/cellranger/rna/feature_ref.py", line 269, in from_transcriptome_and_csv gene_index = cr_reference.NewGeneIndex.load_from_reference(gene_ref_path) File "/mnt/beegfs/users/schugh/cellranger/cellranger-7.0.1/lib/python/cellranger/reference.py", line 657, in load_from_reference raise RuntimeError("couldn't load reference info: " + ensure_str(exc.output)) from exc RuntimeError: couldn't load reference info: thread 'main' panicked at 'assertion failed: (left == right) left: 0, right: 1492: Expected a transcript length of 1492, but got 0 stitching the exons for Transcript { idx: TranscriptIdx( 2706, ), gene_idx: GeneIdx( 667, ), id: "ENSMUST00000160792", chrom: "chr1", strand: Reverse, exons: [ Exon { start: 85088406, end: 85088816, }, Exon { start: 85089526, end: 85089580, }, Exon { start: 85091076, end: 85091189, }, Exon { start: 85094296, end: 85094359, }, Exon { start: 85099476, end: 85099648, }, Exon { start: 85100531, end: 85100694, }, Exon { start: 85109336, end: 85109853, }, ], properties: [ ( "gene_id", "ENSMUSG00000089844", ), ( "gene_version", "7", ), ( "transcript_id", "ENSMUST00000160792", ), ( "transcript_version", "7", ), ( "gene_type", "protein_coding", ), ( "gene_name", "A530032D15Rik", ), ( "transcript_type", "protein_coding", ), ( "transcript_name", "A530032D15Rik-201", ), ( "level", "2", ), ( "protein_id", "ENSMUSP00000125122.1", ), ( "transcript_support_level", "1", ), ( "mgi_id", "MGI:3037746", ), ( "tag", "basic", ), ( "tag", "appris_principal_2", ), ( "tag", "CCDS", ), ( "ccdsid", "CCDS15107.1", ), ( "havana_gene", "OTTMUSG00000029176.35", ), ( "havana_transcript", "OTTMUST00000072362.3", ), ], }', execroot/exec/lib/rust/transcriptome/src/transcriptome.rs:63:9 stack backtrace: 0: rust_begin_unwind at /rustc/9d1b2106e23b1abd32fce1f17267604a5102f57a/library/std/src/panicking.rs:498:5 1: core::panicking::panic_fmt at /rustc/9d1b2106e23b1abd32fce1f17267604a5102f57a/library/core/src/panicking.rs:116:14 2: core::panicking::assert_failed_inner 3: core::panicking::assert_failed at /rustc/9d1b2106e23b1abd32fce1f17267604a5102f57a/library/core/src/panicking.rs:154:5 4: transcriptome::transcriptome::Transcript::get_sequence at execroot/exec/lib/rust/execroot/exec/lib/rust/transcriptome/src/transcriptome.rs:63:9 5: transcriptome::transcriptome::Transcript::gc_content at execroot/exec/lib/rust/execroot/exec/lib/rust/transcriptome/src/transcriptome.rs:84:9 6: transcriptome::python_gene_index::python_gene_index at execroot/exec/lib/rust/execroot/exec/lib/rust/transcriptome/src/python_gene_index.rs:122:26 7: transcriptome::python_gene_index::generate_gene_index at execroot/exec/lib/rust/execroot/exec/lib/rust/transcriptome/src/python_gene_index.rs:217:13 8: transcriptome::python_gene_index::write_gene_index at execroot/exec/lib/rust/execroot/exec/lib/rust/transcriptome/src/python_gene_index.rs:254:13 9: gtf_to_gene_index::main at execroot/exec/lib/rust/transcriptome/src/bin/gtf_to_gene_index.rs:23:15 10: core::ops::function::FnOnce::call_once at /rustc/9d1b2106e23b1abd32fce1f17267604a5102f57a/library/core/src/ops/function.rs:227:5 note: Some details are omitted, run with RUST_BACKTRACE=full for a verbose backtrace.

Waiting 6 seconds for UI to do final refresh. h5py: 3.6.0 pandas: 1.3.5 STAR: 2.7.2a samtools: samtools 1.12 Pipestance failed. Use --noexit option to keep UI running after failure.

2023-09-15 14:54:33 Shutting down. Saving pipestance info to "svi_sample1/svi_sample1.mri.tgz" For assistance, upload this file to 10x Genomics by running:

Any help would be appreciated, thanks

[sagrikachugh]

I tried using version 7.1.0 but still run into error as below

Martian Runtime - v4.0.10 2023-09-15 15:27:58 [jobmngr] WARNING: User-supplied amount 100 GB is higher than the detected cgroup memory limit of 60.3 GB Serving UI at http://scgn01.svi.edu.au:33817?auth=clzUggAlKWhKqu5boKRPNaQQQHzJUfGkTYNkkG_XuFU

Running preflight checks (please wait)... Checking feature definition file... Checking sample info... Checking FASTQ folder... Checking reference... Checking reference_path (/mnt/beegfs/mccarthy/backed_up/general/schugh/Projects/Purton_lab/reference_genome/mm10-new/refdata-gex-mm10-2020-A) on scgn01.svi.edu.au... Checking optional arguments... mro: v4.0.10 mrp: v4.0.10 Anaconda: Python 3.9.7 numpy: 1.22.3 scipy: 1.9.1 pysam: 0.17.0 h5py: 3.6.0

[error] Pipestance failed. Error log at: svi_sample1/SC_RNA_COUNTER_CS/CELLRANGER_PREFLIGHT/fork0/chnk0-u5a2203eb61/_errors

Log message: Traceback (most recent call last): File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/python/cellranger/reference.py", line 656, in load_from_reference subprocess.check_output(cmd, stderr=subprocess.STDOUT) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/anaconda/lib/python3.9/subprocess.py", line 424, in check_output return run(*popenargs, stdout=PIPE, timeout=timeout, check=True, File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/anaconda/lib/python3.9/subprocess.py", line 528, in run raise CalledProcessError(retcode, process.args, subprocess.CalledProcessError: Command '[b'/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/bin/gtf_to_gene_index', b'/mnt/beegfs/mccarthy/backed_up/general/schugh/Projects/Purton_lab/reference_genome/mm10-new/refdata-gex-mm10-2020-A', b'gene_index.json']' returned non-zero exit status 1.

The above exception was the direct cause of the following exception:

Traceback (most recent call last): File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 648, in _main stage.main() File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 607, in main self._run(lambda: self._module.main(args, outs)) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 564, in _run cmd() File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/external/martian/adapters/python/martian_shell.py", line 607, in self._run(lambda: self._module.main(args, outs)) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/mro/rna/stages/common/cellranger_preflight/init.py", line 60, in main run_preflight_checks(args) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/mro/rna/stages/common/cellranger_preflight/init.py", line 34, in run_preflight_checks cr_preflight.check_sample_info( File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/python/cellranger/preflight.py", line 673, in check_sample_info feature_ref = try_load_feature_ref(reference_path, feature_ref_path) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/python/cellranger/preflight.py", line 320, in try_load_feature_ref feature_ref = rna_feature_ref.from_transcriptome_and_csv( File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/python/cellranger/rna/feature_ref.py", line 296, in from_transcriptome_and_csv gene_index = cr_reference.NewGeneIndex.load_from_reference(gene_ref_path) File "/mnt/beegfs/users/schugh/opt/cellranger-7.1.0/lib/python/cellranger/reference.py", line 658, in load_from_reference raise RuntimeError("couldn't load reference info: " + ensure_str(exc.output)) from exc RuntimeError: couldn't load reference info: Bad address (os error 14)

Waiting 6 seconds for UI to do final refresh. Pipestance failed. Use --noexit option to keep UI running after failure.

2023-09-15 15:28:37 Shutting down. 2023-09-15 15:28:37 Caught signal terminated 2023-09-15 15:28:37 [monitor] Caught signal terminated Saving pipestance info to "svi_sample1/svi_sample1.mri.tgz" For assistance, upload this file to 10x Genomics by running:

[evolvedmicrobe]

Hi, please reach out to support@10xgenomics.com, they'll be able to help you and access your uploaded file with diagnostic information.

[sagrikachugh]

I did upload a file earlier and haven't received a response yet, I haven't uploaded any file this time and posted my issue here, do you suggest I should again try uploading my file?

[elehnert10x]

Our records show that a response was sent when you uploaded your file - it may have gone to you spam folder? If you are still having an issue and would like support, please reach out to support@10xgenomics.com.