Question: Hi all, I am fairly new to scRNA-Seq. I am trying to align 10X datasets using cellranger. However, cellranger fails at the mapping step during chemistry detection, and I do not understand why.

This is my code:

cellranger count --id=4CHH4R_tumor1 \
--fastqs=data/K46R-4CHH4R/tumor1-h01 \
--transcriptome=ReferenceGenome/refdata-cellranger-GRCh38-3.0.0/ \
--localcores=12

Running it results in the following error:

/software/cellranger/3.0.1/cellranger-cs/3.0.1/bin
cellranger count (3.0.1)
Copyright (c) 2018 10x Genomics, Inc. All rights reserved.
-------------------------------------------------------------------------------

Martian Runtime - '3.0.1-v3.1.0'
2019-11-19 14:12:15 [jobmngr] WARNING: configured to use 540GB of local memory, but only 180.2GB is currently available.
Serving UI at http://odcf-worker01:40484?auth=MIDdzQZmtbTMaiF1LkOT2qnuVXfoTepZAIa60S11Us0

Running preflight checks (please wait)...
2019-11-19 14:12:16 [runtime] (ready) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.EXPAND_SAMPLE_DEF
2019-11-19 14:12:16 [runtime] (run:local) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.EXPAND_SAMPLE_DEF.fork0.chnk0.main
2019-11-19 14:12:28 [runtime] (chunks_complete) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.EXPAND_SAMPLE_DEF
Checking sample info...
Checking FASTQ folder...
Checking reference...
Checking reference_path (ReferenceGenome/refdata-cellranger-GRCh38-3.0.0/) on odcf-worker01...
Checking chemistry...
Checking optional arguments...
mrc: '3.0.1-v3.1.0'

mrp: '3.0.1-v3.1.0'

Anaconda: Python 2.7.14 :: Anaconda, Inc.

numpy: 1.14.2

scipy: 1.0.1

pysam: 0.14.1

h5py: 2.8.0

pandas: 0.22.0

STAR: STAR_2.5.1b

samtools: samtools 1.7
Using htslib 1.7
Copyright (C) 2018 Genome Research Ltd.

2019-11-19 14:12:50 [runtime] (ready) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY
2019-11-19 14:12:50 [runtime] (run:local) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY.fork0.chnk0.main
2019-11-19 14:12:50 [runtime] (ready) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SC_RNA_ANALYZER.CHOOSE_DIMENSION_REDUCTION
2019-11-19 14:12:50 [runtime] (run:local) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SC_RNA_ANALYZER.CHOOSE_DIMENSION_REDUCTION.fork0.chnk0.main
2019-11-19 14:12:50 [runtime] (ready) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.DISABLE_FEATURE_STAGES
2019-11-19 14:12:50 [runtime] (run:local) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.DISABLE_FEATURE_STAGES.fork0.chnk0.main
2019-11-19 14:12:53 [runtime] (chunks_complete) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.DISABLE_FEATURE_STAGES
2019-11-19 14:12:53 [runtime] (chunks_complete) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.SC_RNA_ANALYZER.CHOOSE_DIMENSION_REDUCTION
2019-11-19 14:13:02 [runtime] (progress) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY.fork0.chnk0: Building transcriptome...
2019-11-19 14:18:16 [runtime] (progress) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY.fork0.chnk0: Building kmer index...
2019-11-19 14:19:47 [runtime] (progress) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY.fork0.chnk0: Mapping reads...
2019-11-19 14:19:51 [runtime] (failed) ID.4CHH4R_tumor1.SC_RNA_COUNTER_CS.SC_RNA_COUNTER.CHEMISTRY_DETECTOR.DETECT_CHEMISTRY

[error] Pipestance failed. Error log at:
4CHH4R_tumor1/SC_RNA_COUNTER_CS/SC_RNA_COUNTER/CHEMISTRY_DETECTOR/DETECT_CHEMISTRY/fork0/chnk0-u55c3d3ea52/_errors

Log message:
Traceback (most recent call last):
File "/software/cellranger/3.0.1/martian-cs/v3.1.0/adapters/python/martian_shell.py", line 554, in _main
stage.main()
File "/software/cellranger/3.0.1/martian-cs/v3.1.0/adapters/python/martian_shell.py", line 519, in main
self._run(lambda: self._module.main(args, outs))
File "/software/cellranger/3.0.1/martian-cs/v3.1.0/adapters/python/martian_shell.py", line 488, in _run
cmd()
File "/software/cellranger/3.0.1/martian-cs/v3.1.0/adapters/python/martian_shell.py", line 519, in
self._run(lambda: self._module.main(args, outs))
File "/software/cellranger/3.0.1/cellranger-cs/3.0.1/mro/stages/chemistry_detector/detect_chemistry/__init__.py", line 348, in main
chemistry_name, _report, metrics = infer_sc3p_or_sc5p(chunks, txome_idx, vdj_idx)
File "/software/cellranger/3.0.1/cellranger-cs/3.0.1/mro/stages/chemistry_detector/detect_chemistry/__init__.py", line 218, in infer_sc3p_or_sc5p
metrics[read_type] = map_reads(fq_specs[read_type], kmer_idx_path, map_out_path)
File "/software/cellranger/3.0.1/cellranger-cs/3.0.1/mro/stages/chemistry_detector/detect_chemistry/__init__.py", line 91, in map_reads
'--fastqs', fq_spec_path])
File "/software/cellranger/3.0.1/cellranger-cs/3.0.1/mro/stages/chemistry_detector/detect_chemistry/__init__.py", line 85, in run
tk_subproc.check_call(args)
File "/software/cellranger/3.0.1/cellranger-cs/3.0.1/tenkit/lib/python/tenkit/log_subprocess.py", line 37, in check_call
return subprocess.check_call(*args, **kwargs)
File "/software/cellranger/3.0.1/miniconda-cr-cs/4.3.21-miniconda-cr-cs-c10/lib/python2.7/subprocess.py", line 186, in check_call
raise CalledProcessError(retcode, cmd)
CalledProcessError: Command '['detect_chemistry', 'map-reads', '4CHH4R_tumor1/SC_RNA_COUNTER_CS/SC_RNA_COUNTER/CHEMISTRY_DETECTOR/DETECT_CHEMISTRY/fork0/chnk0-u55c3d3ea52/files/kmers.idx', '/dev/null', '4CHH4R_tumor1/SC_RNA_COUNTER_CS/SC_RNA_COUNTER/CHEMISTRY_DETECTOR/DETECT_CHEMISTRY/fork0/chnk0-u55c3d3ea52/files/R1_out.json', '--initial-reads', '10000', '--fastqs', '4CHH4R_tumor1/SC_RNA_COUNTER_CS/SC_RNA_COUNTER/CHEMISTRY_DETECTOR/DETECT_CHEMISTRY/fork0/chnk0-u55c3d3ea52/files/R1_in.json']' returned non-zero exit status 101

Can anybody give me an idea what is going wrong here?

I downloaded the reference genome from the 10X genomics website.

Answer: I think I solved the issue now. I realized that the directory specified in `--fastqs` only contained symlinks to the acutal fastq files, which cellranger apparently could not handle. Why it failed only at that specific step I do not know, but it runs smoothly now that I specified the correct directory with the fastq files.