Cellranger count argument for 10xgenomic RNA+surface protein

Question

[Stemcellguy]

I can't get cellranger count work. Dataset from here: https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.0/pbmc_1k_protein_v3. The datasets has two FASTQ files, one for RNA and another for surface protein.

Here is what I did:

1- downloaded the FASTQ file, cellranger 3 and the reference genome GRCh38-3.0.0 into one directory in cluster computer 2- untag the FASTQ files. 3- ran the argument for the RNA FASTQ first:

cellranger-3.0.2/cellranger count --id=pbmc_1k_protein_v3_fastqs --transcriptome=refdata-cellranger-GRCh38-3.0.0 --fastqs=path/pbmc_1k_protein_v3_fastqs/pbmc_1k_protein_v3_gex_fastqs/ —sample=pbmc_1k_protein_v3_fastqs --localcores=40 --expect-cells=1000 --localmem=200

I don't get error, but the cellranger doesn't run!

[swbarnes2]

Did you try cellranger count --help ?

[Stemcellguy]

Yes, I didn't see any problem with my argument

[ATpoint]

Doesn't run means what exactly?

[Stemcellguy]

It doesn't give errors but nothing happens and it takes me back to the command line. It only shows this:

cellranger count (3.0.2)
Copyright (c) 2019 10x Genomics, Inc.  All rights reserved.
-------------------------------------------------------------------------------
Usage:
    count
        --id=ID
        [--fastqs=PATH]
        [--sample=PREFIX]
        --transcriptome=DIR
        [options]
    count <run_id> <mro> [options]
    count -h | --help | --version

[emily.dabe]

I am running into the exact same problem. Test run was successful etc. @ATpoint did you ever figure out the issue?

[Friederike]

seems to me that sample needs 2 hyphens, too:

--sample=mysample

Answer 1

[Arup Ghosh]

1) Have you verified the Cell Ranger installation?

Verify Installation

2) The --id option will be used to create the output directory (also for the library id) so make sure to keep the fastq files in a different directory than the current working directory or use a different id. You are using pbmc_1k_protein_v3_fastqs for id option as well as the raw file.

3) If 2 is not the case check the current working directory is there is a new pbmc_1k_protein_v3_fastqs created and share the contents of _log file.

[Stemcellguy]

1- Yes, test run was successful.

2- I tried different --id, same result. It does't create any output.

Please take a look at my directory (I created the pbmc1k_output before running the code):

    ls -1 path 
    cellranger-3.0.2
    pbmc_1k_protein_v3_fastqs
    pbmc1k_output
    refdata-cellranger-GRCh38-3.0.0
    testrun

    ls -1 cellranger-3.0.2
    builtwith.json
    cellranger
    cellranger-cs
    cellranger-shell
    cellranger-tiny-fastq
    cellranger-tiny-ref
    lz4
    martian-cs
    miniconda-cr-cs
    product.json
    sourceme.bash
    sourceme.csh
    STAR

    ls -1  pbmc_1k_protein_v3_fastqs
    pbmc_1k_protein_v3_gex_fastqs
    pbmc_1k_protein_v3_antibody_fastqs

    ls -1  pbmc_1k_protein_v3_gex_fastqs
    pbmc_1k_protein_v3_gex_S1_L001_I1_001.fastq
    pbmc_1k_protein_v3_gex_S1_L001_R1_001.fastq
    pbmc_1k_protein_v3_gex_S1_L001_R2_001.fastq
    pbmc_1k_protein_v3_gex_S1_L002_I1_001.fastq
    pbmc_1k_protein_v3_gex_S1_L002_R1_001.fastq
    pbmc_1k_protein_v3_gex_S1_L002_R2_001.fastq

    ls -1 pbmc_1k_protein_v3_antibody_fastqs
    pbmc_1k_protein_v3_antibody_S2_L001_I1_001.fastq
    pbmc_1k_protein_v3_antibody_S2_L001_R1_001.fastq
    pbmc_1k_protein_v3_antibody_S2_L001_R2_001.fastq
    pbmc_1k_protein_v3_antibody_S2_L002_I1_001.fastq
    pbmc_1k_protein_v3_antibody_S2_L002_R1_001.fastq
    pbmc_1k_protein_v3_antibody_S2_L002_R2_001.fastq

Now the code I ran again was:

cellranger-3.0.2/cellranger count --id=/path/pbmc1k_output--transcriptome=refdata-cellranger-GRCh38-3.0.0 --fastqs=/path/pbmc_1k_protein_v3_fastqs/--sample=pbmc_1k --localcores=40 --expect-cells=1000 --localmem=200

Answer 2

[emily.dabe]

Okay I figured it out for mine I think. So the "samplename" portion of the fastq can't have underscores. I changed mine to TestSampleA8_S3_L001_R2_001.fastq.gz instead of a longer underscore separated name and its working now. Let me know if that works for you.

[swbarnes2]

If the sample fastqs given by 10xGenomics have underscores, then underscores have to be fine.

Answer 3

[swbarnes2]

How is the software supposed to understand which sample you want to look at with "pbmc1k"? There are no samples in there with names like pbmc1K_S1_L001...

Try --sample=pbmc_1k_protein_v3_gex