Question: Cellranger count argument for 10xgenomic RNA+surface protein
0
gravatar for Stemcellguy
13 months ago by
Stemcellguy 0 wrote:

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!

rna-seq • 1.2k views
ADD COMMENTlink modified 12 months ago by swbarnes27.8k • written 13 months ago by Stemcellguy 0

Did you try cellranger count --help ?

ADD REPLYlink written 13 months ago by swbarnes27.8k

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

ADD REPLYlink written 13 months ago by Stemcellguy 0

Doesn't run means what exactly?

ADD REPLYlink written 13 months ago by ATpoint35k

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
ADD REPLYlink modified 13 months ago • written 13 months ago by Stemcellguy 0

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

ADD REPLYlink written 12 months ago by emily.dabe0

seems to me that sample needs 2 hyphens, too:

--sample=mysample
ADD REPLYlink written 12 months ago by Friederike5.7k
3
gravatar for Arup Ghosh
13 months ago by
Arup Ghosh2.4k
India
Arup Ghosh2.4k wrote:

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.

ADD COMMENTlink modified 13 months ago • written 13 months ago by Arup Ghosh2.4k

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
ADD REPLYlink modified 13 months ago • written 13 months ago by Stemcellguy 0
0
gravatar for emily.dabe
12 months ago by
emily.dabe0
emily.dabe0 wrote:

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.

ADD COMMENTlink written 12 months ago by emily.dabe0

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

ADD REPLYlink written 12 months ago by swbarnes27.8k
0
gravatar for swbarnes2
12 months ago by
swbarnes27.8k
United States
swbarnes27.8k wrote:

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

ADD COMMENTlink modified 12 months ago • written 12 months ago by swbarnes27.8k
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