Cellranger count pipestance failed: The read lengths are incompatible with all the chemistries

Question

[firestar]

Hi,

I have been given some FastQ files from Illumina MiSeq containing 10X single-cell gene expression data. When I run CellRanger, I get the following error:

Log message:
The read lengths are incompatible with all the chemistries for Sample GEX083 in "/crex/proj/snic2022-22-123/nobackup/roy/tonsil/data/raw/Fastq".
- read1 median length = 28
- read2 median length = 0
- index1 median length = 0

The minimum read length for different chemistries are:
SC5P-R2 - read1: 26, read2: 25, index1: 0
SC5P-PE - read1: 81, read2: 25, index1: 0
SC3Pv1 - read1: 25, read2: 10, index1: 14
SC3Pv2 - read1: 26, read2: 25, index1: 0
SC3Pv3 - read1: 26, read2: 15, index1: 0
SC3Pv3LT - read1: 26, read2: 25, index1: 0

We expect that at least 50% of the reads exceed the minimum length.

Any ideas on what might be wrong?

---

More details

I have the following files at data/raw/Fastq/:

FastqSummaryF1L1.txt
FastqSummaryF1L3.txt
FastqSummaryF1L2.txt
FastqSummaryF1L4.txt
GEX083_S1_L001_R1_001.fastq.gz
GEX083_S1_L003_R1_001.fastq.gz
GEX083_S1_L002_R1_001.fastq.gz

---

Heads of the three fastq files are shown here:

zcat GEX083_S1_L001_R1_001.fastq.gz | head

@NB501805:156:HCW2WBGXK:1:11101:8290:1042 1:N:0:1
TCCCANGCATACAGCTTCGCGCCGTTGT
+
AAAAA#EEEEEEEEEEEEEEEEEEEEEE
@NB501805:156:HCW2WBGXK:1:11101:3197:1042 1:N:0:1
AGCTCNAGTCTCTCCACGGACGCGATAT
+
AAAAA#EEEEEEEEEEEEEEEEEEEEEE
@NB501805:156:HCW2WBGXK:1:11101:19026:1042 1:N:0:1
ATGGGNGCACGCCACATGGCAGGCCCGC


zcat GEX083_S1_L002_R1_001.fastq.gz | head

@NB501805:156:HCW2WBGXK:2:11101:18093:1042 1:N:0:1
CTGATCCCAGGTGTTTATGTGGCCNTCC
+
AAAAAEEEEEEEEEEEEEEEEEEE#EEA
@NB501805:156:HCW2WBGXK:2:11101:2744:1042 1:N:0:1
GTCCTCACATTGCCGGAGTTGAGCNGGA
+
AAAAAEEEEEEEEEEEEEEEE/EE#EEE
@NB501805:156:HCW2WBGXK:2:11101:5237:1042 1:N:0:1
ATAGGCTAGCATTGAAGCCCCTAGNCAG


zcat GEX083_S1_L003_R1_001.fastq.gz | head

@NB501805:156:HCW2WBGXK:3:11401:25091:1021 1:N:0:1
AAANCCACATGGCTATTTTTCATCCTGT
+
AAA#AEEEEAAEEEEEEEEEE/EE/EAE
@NB501805:156:HCW2WBGXK:3:11401:5497:1021 1:N:0:1
TTGNCTGCAGTTGTCACCATGCCCGGCT
+
AAA#AEEEEEEEEEEEEEEEEEEEEEEE
@NB501805:156:HCW2WBGXK:3:11401:21897:1021 1:N:0:1
AGTAGCTTCTGCCCTAAATGTAGAATAT

---

And I am running cellranger count as follows:

module load bioinfo-tools
module load cellranger/6.0.2

id="GEX083"
path="data/raw/Fastq/"
transcriptome="${CELLRANGER_DATA}/refdata-gex-GRCh38-2020-A/"

cellranger count \
--nosecondary \
--id $id \
--transcriptome=${transcriptome} \
--fastqs $path \
--sample $id \
--localcores 20 \
--localmem 128

---

Full run log is given below:

Martian Runtime - v4.0.4
2022-06-21 22:00:43 [jobmngr] WARNING: User-supplied amount 128 GB is higher than the detected cgroup memory limit of 123.1 GB
2022-06-21 22:00:43 [jobmngr] WARNING: configured to use 128GB of local memory, but only 105.2GB is currently available.
2022-06-21 22:00:43 [jobmngr] WARNING: detected a cgroup soft memory limit of 123.1GB. If the system runs low on memory, jobs may get killed.
Serving UI at http://r409.uppmax.uu.se:42792?auth=dhbMqhur8mSlD8So2y5KzeKhrnjTIhbmYJn81H9OcAY

Running preflight checks (please wait)...
Checking sample info...
Checking FASTQ folder...
Checking reference...
Checking reference_path (/crex/data/Chromium/cellranger-data/2020-A/refdata-gex-GRCh38-2020-A) on r409.uppmax.uu.se...
Checking optional arguments...
mrc: v4.0.4

mrp: v4.0.4

Anaconda: Python 3.7.6

numpy: 1.19.2

scipy: 1.1.0

pysam: 0.16.0.1

h5py: 2.8.0

pandas: 0.24.2

STAR: 2.7.2a

samtools: samtools 1.10
Using htslib 1.10.2
Copyright (C) 2019 Genome Research Ltd.

2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_CHEMISTRY_DETECTOR._GEM_WELL_CHEMISTRY_DETECTOR.DETECT_COUNT_CHEMISTRY
2022-06-21 22:00:51 [runtime] (run:local) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_CHEMISTRY_DETECTOR._GEM_WELL_CHEMISTRY_DETECTOR.DETECT_COUNT_CHEMISTRY.fork0.chnk0.main
2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.WRITE_GENE_INDEX
2022-06-21 22:00:51 [runtime] (run:local) ID.GEX083.SC_RNA_COUNTER_CS.WRITE_GENE_INDEX.fork0.chnk0.main
2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.SANITIZE_MAP_CALLS
2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.COUNT_GEM_WELL_PROCESSOR._BASIC_SC_RNA_COUNTER.DISABLE_BAMS
2022-06-21 22:00:51 [runtime] (run:local) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.COUNT_GEM_WELL_PROCESSOR._BASIC_SC_RNA_COUNTER.DISABLE_BAMS.fork0.chnk0.main
2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MAKE_FULL_CONFIG._MAKE_VDJ_CONFIG
2022-06-21 22:00:51 [runtime] (run:local) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MAKE_FULL_CONFIG._MAKE_VDJ_CONFIG.fork0.chnk0.main
2022-06-21 22:00:51 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.FULL_COUNT_INPUTS.WRITE_GENE_INDEX
2022-06-21 22:00:51 [runtime] (run:local) ID.GEX083.SC_RNA_COUNTER_CS.FULL_COUNT_INPUTS.WRITE_GENE_INDEX.fork0.chnk0.main
2022-06-21 22:00:52 [runtime] (chunks_complete) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MAKE_FULL_CONFIG._MAKE_VDJ_CONFIG
2022-06-21 22:00:52 [runtime] (chunks_complete) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.COUNT_GEM_WELL_PROCESSOR._BASIC_SC_RNA_COUNTER.DISABLE_BAMS
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.MULTI_SETUP_CHUNKS
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.MULTI_SETUP_CHUNKS
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.MAKE_SHARD
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.MAKE_SHARD
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.BARCODE_CORRECTION
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.BARCODE_CORRECTION
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.RUST_BRIDGE
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.ASSEMBLE_VDJ
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.RUST_BRIDGE
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.ASSEMBLE_VDJ
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_T_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.MERGE_METRICS
2022-06-21 22:00:52 [runtime] (ready) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_GEM_WELL_PROCESSOR.VDJ_B_GEM_WELL_PROCESSOR.SC_VDJ_CONTIG_ASSEMBLER.MERGE_METRICS
2022-06-21 22:00:53 [runtime] (failed) ID.GEX083.SC_RNA_COUNTER_CS.SC_MULTI_CORE.MULTI_CHEMISTRY_DETECTOR._GEM_WELL_CHEMISTRY_DETECTOR.DETECT_COUNT_CHEMISTRY

[error] Pipestance failed. Error log at:
GEX083/SC_RNA_COUNTER_CS/SC_MULTI_CORE/MULTI_CHEMISTRY_DETECTOR/_GEM_WELL_CHEMISTRY_DETECTOR/DETECT_COUNT_CHEMISTRY/fork0/chnk0-u7fb5b22373/_errors

Log message:
The read lengths are incompatible with all the chemistries for Sample GEX083 in "/crex/proj/snic2022-22-123/nobackup/roy/tonsil/data/raw/Fastq".
- read1 median length = 28
- read2 median length = 0
- index1 median length = 0

The minimum read length for different chemistries are:
SC5P-R2 - read1: 26, read2: 25, index1: 0
SC5P-PE - read1: 81, read2: 25, index1: 0
SC3Pv1 - read1: 25, read2: 10, index1: 14
SC3Pv2 - read1: 26, read2: 25, index1: 0
SC3Pv3 - read1: 26, read2: 15, index1: 0
SC3Pv3LT - read1: 26, read2: 25, index1: 0

We expect that at least 50% of the reads exceed the minimum length.


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

[biofalconch]

Hi, it seems you only have one of the reads (read 1) coming from different lanes of the sequencer (thus the L001-L003), do you have any other files?

[firestar]

These are all the file I have received from a colleague. I was also wondering if some files were missing, but assumed it was perhaps just single-end reads. I could ask again to double check.

[1363390017]

I also encountered the same problem, is single-end sequencing unable to analyze?

[songyy199802]

hi, have you solved this problem? I have the same problem. Excuse me, what is the problem and how to solve it? and, How did you solve it at last? thanks!

[ATpoint]

The problem is described in my comments, OP had single-end reads which makes no sense. 10x Chromium is a paired-end sequencing. For more help please open a new question with details on what commands you run and what the errors are.

Answer 1

[ATpoint]

Yes, as in any 10x R1 contains UMI+CB and R2 the cDNA. Single-end makes no sense.

[GenoMax]

To elaborate further on this answer. R1 file examples shown in the original post are 26 bp UMI+Cell barcode reads. R2 files is where you will find the actual cDNA (~50 bp) read. There may also be a I1 file for the Illumina index sequences. This file is optional and may not always be there.

So if you just get a R1 or R2 file that data is not usable at all.