Open GGN1999 opened 2 weeks ago
As the message shown, the reads mapping to vdj gene is very low, you need check the sequencing data before analyzing. To overcome this error on the command line, set the --chain parameter to either IG for BCRs or TR for TCRs.
As the message shown, the reads mapping to vdj gene is very low, you need check the sequencing data before analyzing. To overcome this error on the command line, set the --chain parameter to either IG for BCRs or TR for TCRs.
solution
- cellranger 6.1.2 is fine. update celescope to 1.16.1
- add --other_param " --chain=IG " in shell script.
Thank you very much for your reply. I updated celescope to 1.16.1 and used flv_CR to run cellranger vdj on my GEXSCOPE® BCR sequencing data. -other_param " --chain=IG " was also added in shell script. However, I was again encountered with very similar error that said:
2024-08-29 15:14:08 [runtime] (failed) ID.PTCL_50_SHM-B-full_len_vdj.SC_VDJ_ASSEMBLER_CS.SC_MULTI_CORE.MULTI_CHEMISTRY_DETECTOR._GEM_WELL_CHEMISTRY_DETECTOR.VDJ_CHEMISTRY_DETECTOR.DETECT_CHEMISTRY
[error] Pipestance failed. Error log at: PTCL_50_SHM-B-full_len_vdj/SC_VDJ_ASSEMBLER_CS/SC_MULTI_CORE/MULTI_CHEMISTRY_DETECTOR/_GEM_WELL_CHEMISTRY_DETECTOR/VDJ_CHEMISTRY_DETECTOR/DETECT_CHEMISTRY/fork0/chnk0-u0e23d01fbf/_errors
Log message: There were not enough reads to auto detect the chemistry: Sample PTCL_50_SHM-B-full_len_vdj in "/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/PTCL_50_shihuiming/multi_flv_CR/shell/PTCL_50_SHM-B-full_len_vdj/02.convert" Note that you can avoid auto detection by specifying the specific chemistry type and version.
Any possible solution to this error?
This error might be caused by auto-detect chemistry step and the Valid Reads metric is abnormal. Could you provide the whole running message like following screenshot?
the whole running message: 2024-08-29 16:32:06,184 - celescope.tools.sample.sample - INFO - start... Args: Namespace(subparser_assay='flv_CR', outdir='.//PTCL_50_SHM-B-full_len_vdj/00.sample', sample='PTCL_50_SHM-B-full_len_vdj', thread='16', debug=False, fq1='/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz', chemistry='auto', func=<function sample at 0x7f81673cd1f0>) 2024-08-29 16:32:06,187 - celescope.tools.sample.run - INFO - start... 2024-08-29 16:32:06,408 - celescope.tools.barcode.check_chemistry - INFO - start... 2024-08-29 16:32:06,409 - celescope.tools.barcode.check_chemistry - INFO - /dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz 2024-08-29 16:32:06,409 - celescope.tools.barcode.get_chemistry - INFO - start... 2024-08-29 16:32:06,568 - celescope.tools.barcode.get_chemistry - INFO - [('scopeV2.2.1', 8872), ('scopeV2.1.1', 1873), ('flv_rna', 462), ('scopeV2.0.1', 4)] 2024-08-29 16:32:06,568 - celescope.tools.barcode.get_chemistry - INFO - chemistry: scopeV2.2.1 2024-08-29 16:32:06,568 - celescope.tools.barcode.get_chemistry - INFO - done. time used: 0:00:00.159119 2024-08-29 16:32:06,568 - celescope.tools.barcode.check_chemistry - INFO - done. time used: 0:00:00.159411 Sample ID: PTCL_50_SHM-B-full_len_vdj Assay: flv_CR Chemistry: scopeV2.2.1 (kit V1) Software Version: 1.16.1 2024-08-29 16:32:06,574 - celescope.tools.sample.run - INFO - done. time used: 0:00:00.386621 2024-08-29 16:32:06,574 - celescope.tools.step._clean_up - INFO - start... 2024-08-29 16:32:06,575 - celescope.tools.step._render_html - INFO - start... 2024-08-29 16:32:07,049 - celescope.tools.step._render_html - INFO - done. time used: 0:00:00.474010 2024-08-29 16:32:07,049 - celescope.tools.step._clean_up - INFO - done. time used: 0:00:00.475155 2024-08-29 16:32:07,049 - celescope.tools.sample.sample - INFO - done. time used: 0:00:00.864817 2024-08-29 16:32:09,115 - celescope.tools.barcode.barcode - INFO - start... Args: Namespace(subparser_assay='flv_CR', chemistry='auto', pattern=None, whitelist=None, linker=None, lowQual=0, lowNum=2, nopolyT=False, noLinker=False, filterNoPolyT=False, allowNoLinker=False, gzip=False, output_R1=False, fq1='/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz', fq2='/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_2.fq.gz', match_dir=None, stdout=False, outdir='.//PTCL_50_SHM-B-full_len_vdj/01.barcode', sample='PTCL_50_SHM-B-full_len_vdj', thread='16', debug=False, func=<function barcode at 0x7f69143a3ee0>) 2024-08-29 16:32:09,334 - celescope.tools.barcode.check_chemistry - INFO - start... 2024-08-29 16:32:09,334 - celescope.tools.barcode.check_chemistry - INFO - /dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz 2024-08-29 16:32:09,334 - celescope.tools.barcode.get_chemistry - INFO - start... 2024-08-29 16:32:09,397 - celescope.tools.barcode.get_chemistry - INFO - [('scopeV2.2.1', 8872), ('scopeV2.1.1', 1873), ('flv_rna', 462), ('scopeV2.0.1', 4)] 2024-08-29 16:32:09,397 - celescope.tools.barcode.get_chemistry - INFO - chemistry: scopeV2.2.1 2024-08-29 16:32:09,397 - celescope.tools.barcode.get_chemistry - INFO - done. time used: 0:00:00.062198 2024-08-29 16:32:09,397 - celescope.tools.barcode.check_chemistry - INFO - done. time used: 0:00:00.062389 2024-08-29 16:32:09,401 - celescope.tools.barcode.run - INFO - start... 2024-08-29 16:40:48,945 - celescope.tools.barcode.run - INFO - /dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz finished. 2024-08-29 16:40:48,951 - celescope.tools.barcode.add_step_metrics - INFO - start... Raw Reads: 42,425,066 Valid Reads: 4,474(0.01%) Q30 of Barcodes: 96.19% Q30 of UMIs: 96.81% No PolyT Reads: 0(0.0%) Low Quality Reads: 0(0.0%) No Linker Reads: 2,712,399(6.39%) No Barcode Reads: 39,708,193(93.6%) Corrected Linker Reads: 3,041,237(7.17%) Corrected Barcode Reads: 4,473(0.01%) 2024-08-29 16:40:48,952 - celescope.tools.barcode.add_step_metrics - INFO - done. time used: 0:00:00.000255 2024-08-29 16:40:48,953 - celescope.tools.barcode.run - INFO - done. time used: 0:08:39.550611 2024-08-29 16:40:48,953 - celescope.tools.step._clean_up - INFO - start... 2024-08-29 16:40:48,954 - celescope.tools.step._render_html - INFO - start... 2024-08-29 16:40:49,281 - celescope.tools.step._render_html - INFO - done. time used: 0:00:00.326443 2024-08-29 16:40:49,281 - celescope.tools.step._clean_up - INFO - done. time used: 0:00:00.327964 2024-08-29 16:40:49,281 - celescope.tools.barcode.barcode - INFO - done. time used: 0:08:40.165687 Args: Namespace(subparser_assay='flv_CR', soft_path='/dssg/home/acct-medzwli/medzwli_qyr/software/cellranger/cellranger-7.0.1/cellranger', tenX_chemistry='V2', outdir='.//PTCL_50_SHM-B-full_len_vdj/02.convert', sample='PTCL_50_SHM-B-full_len_vdj', thread='16', debug=False, fq2='.//PTCL_50_SHM-B-full_len_vdj/01.barcode/PTCL_50_SHM-B-full_len_vdj_2.fq', func=<function convert at 0x7fa08f6f19d0>) 2024-08-29 16:40:51,644 - celescope.flv_CR.convert.gen_sgr_tenX_dict - INFO - start... 2024-08-29 16:40:51,651 - celescope.flv_CR.convert.gen_sgr_tenX_dict - INFO - done. time used: 0:00:00.006230 2024-08-29 16:40:51,651 - celescope.flv_CR.convert.write_fq1 - INFO - start... 2024-08-29 16:40:51,712 - celescope.flv_CR.convert.write_fq1 - INFO - done. time used: 0:00:00.060868 2024-08-29 16:40:51,712 - celescope.flv_CR.convert.gzip_fq2 - INFO - start... 2024-08-29 16:40:51,751 - celescope.flv_CR.convert.gzip_fq2 - INFO - done. time used: 0:00:00.039630 2024-08-29 16:40:51,752 - celescope.flv_CR.convert.dump_tenX_sgr_barcode_json - INFO - start... 2024-08-29 16:40:51,752 - celescope.tools.utils.dump_dict_to_json - INFO - start... 2024-08-29 16:40:51,752 - celescope.tools.utils.dump_dict_to_json - INFO - done. time used: 0:00:00.000277 2024-08-29 16:40:51,752 - celescope.flv_CR.convert.dump_tenX_sgr_barcode_json - INFO - done. time used: 0:00:00.000371 2024-08-29 16:40:51,752 - celescope.tools.step._clean_up - INFO - start... 2024-08-29 16:40:51,752 - celescope.tools.step._render_html - INFO - start... 2024-08-29 16:40:52,087 - celescope.tools.step._render_html - INFO - done. time used: 0:00:00.334438 2024-08-29 16:40:52,087 - celescope.tools.step._clean_up - INFO - done. time used: 0:00:00.335095 Args: Namespace(subparser_assay='flv_CR', ref_path='/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/refdata-cellranger-vdj-GRCh38-alts-ensembl-7.0.0', soft_path='/dssg/home/acct-medzwli/medzwli_qyr/software/cellranger/cellranger-7.0.1/cellranger', other_param=' --chain=IG ', mem='10', seqtype='BCR', not_refine=False, coeff=1.5, outdir='.//PTCL_50_SHM-B-full_len_vdj/03.assemble', sample='PTCL_50_SHM-B-full_len_vdj', thread='16', debug=False, fqs_dir='.//PTCL_50_SHM-B-full_len_vdj/02.convert', func=<function assemble at 0x7fa27598d280>) 2024-08-29 16:40:54,183 - celescope.flv_CR.assemble.assemble - INFO - start... 2024-08-29 16:40:54,183 - celescope.flv_CR.assemble.assemble - INFO - /dssg/home/acct-medzwli/medzwli_qyr/software/cellranger/cellranger-7.0.1/cellranger vdj --id=PTCL_50_SHM-B-full_len_vdj --reference=/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/refdata-cellranger-vdj-GRCh38-alts-ensembl-7.0.0 --fastqs=/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/multi_flv_CR/shell/PTCL_50_SHM-B-full_len_vdj/02.convert --sample=PTCL_50_SHM-B-full_len_vdj --localcores=16 --localmem=10 --chain=IG 2024-08-29 16:41:23,335 - celescope.tools.step._clean_up - INFO - start... Traceback (most recent call last): File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/lib/python3.9/site-packages/celescope/flv_CR/assemble.py", line 79, in assemble runner.run() File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/lib/python3.9/site-packages/celescope/flv_CR/assemble.py", line 72, in run self.assemble() File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/lib/python3.9/site-packages/celescope/tools/utils.py", line 45, in wrapper result = func(*args, **kwargs) File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/lib/python3.9/site-packages/celescope/flv_CR/assemble.py", line 67, in assemble subprocess.check_call(cmd, shell=True) File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/lib/python3.9/subprocess.py", line 373, in check_call raise CalledProcessError(retcode, cmd) subprocess.CalledProcessError: Command '/dssg/home/acct-medzwli/medzwli_qyr/software/cellranger/cellranger-7.0.1/cellranger vdj --id=PTCL_50_SHM-B-full_len_vdj --reference=/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/refdata-cellranger-vdj-GRCh38-alts-ensembl-7.0.0 --fastqs=/dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/multi_flv_CR/shell/PTCL_50_SHM-B-full_len_vdj/02.convert --sample=PTCL_50_SHM-B-full_len_vdj --localcores=16 --localmem=10 --chain=IG ' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/dssg/home/acct-medzwli/medzwli_qyr/.conda/envs/convert_10X/bin/celescope", line 8, in
Valid Reads metric is abnormal. You need check the fastqs whether is single-cell full length VDJ sequencing data. Running message means your input fastqs is auto detected to be "scopeV2.2.1" which is single-cell RNA-Seq sequencing data.
2024-08-29 16:32:06,408 - celescope.tools.barcode.check_chemistry - INFO - start...
2024-08-29 16:32:06,409 - celescope.tools.barcode.check_chemistry - INFO - /dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz
2024-08-29 16:32:06,409 - celescope.tools.barcode.get_chemistry - INFO - start...
2024-08-29 16:32:06,568 - celescope.tools.barcode.get_chemistry - INFO - [('scopeV2.2.1', 8872), ('scopeV2.1.1', 1873), ('flv_rna', 462), ('scopeV2.0.1', 4)]
2024-08-29 16:32:06,568 - celescope.tools.barcode.get_chemistry - INFO - chemistry: scopeV2.2.1
2024-08-29 16:32:09,401 - celescope.tools.barcode.run - INFO - start...
2024-08-29 16:40:48,945 - celescope.tools.barcode.run - INFO - /dssg/home/acct-medzwli/medzwli_qyr/PTCL_single_cell/2023Ruiang/healthy_donor/PTCL_50_shihuiming/20231117_E150016801_U1119_DXB90-3/20231117_E150016801_U1119_DXB90-3/DXB90-3/E150016801_L01_DXB90-3_1.fq.gz finished.
2024-08-29 16:40:48,951 - celescope.tools.barcode.add_step_metrics - INFO - start...
Raw Reads: 42,425,066
Valid Reads: 4,474(0.01%)
I attempted to analyze BCR sequencing data generated using the GEXSCOPE® Single-Cell Immune Receptor Library Construction Kit with the multi_vdj_full_len tool. However, I encountered an error during the 03.assemble step.
Error Message:
V(D)J Chain detection failed for Sample xxx-full_len_vdj in "xxx/02.convert".
Total Reads = 1000000 Reads mapped to TR = 4 Reads mapped to IG = 142
In order to distinguish between the TR and the IG chain the following conditions need to be satisfied:
Are there any specific adjustments or parameters I can use to bypass or correct this error? Or is there another way to obtain filtered_contig.fasta and filtered_contig_annotations.csv files that are compatible with cellranger vdj outputs for subsequent BCR analysis using Change-O?
Version: celescope 1.8.1 cellranger 6.1.2
Thank you!