single cell - generating cellranger counts from FASTQ files

Question

[shivangi.agarwal800]

Hi Guys,

I am trying to run cellranger count go generate count matrix for the sequenced fastq on NextSeq 500. My fastqs are named like:

SRR12345671.fastq.gz 
SRR12345672.fastq.gz 
SRR12345673.fastq.gz 
SRR12345674.fastq.gz 
SRR12345675.fastq.gz 

I am using following command:

cellranger count --id=SRR1234567 --fastqs=/home/user/cellranger_count/fastqs
--sample=SRR1234567  --transcriptome=/mnt/home/user/run-cellranger/refdata-cellranger-GRCh38-3.0.0

but getting the following error as:

ERROR: No input FASTQs were found for the requested parameters.

If your files came from bcl2fastq or mkfastq:

• Make sure you are specifying the correct --sample(s), i.e. matching the sample sheet
• Make sure your files follow the correct naming convention, e.g. SampleName_S1_L001_R1_001.fastq.gz (and the R2 version)
• Make sure your --fastqs points to the correct location.
• Make sure your --lanes, if any, are correctly specified.

Refer to the "Specifying Input FASTQs" page at https://support.10xgenomics.com/ for more details.

Please help me to fix this.

Answer 1

[GenoMax]

See the last section here: https://support.10xgenomics.com/de-novo-assembly/software/pipelines/latest/using/fastq-input

You need to rename your files so they follow a convention cellranger expects.

[Sample Name]_S1_L00[Lane Number]_[Read Type]_001.fastq.gz

Where Read Type is one of:

    I1: Sample index read (optional)
    I2: Sample index read (optional)
    R1: Read 1
    R2: Read 2

[shivangi.agarwal800]

Hi,

Thanks. But, I see did not get the fastqs as R1, R2 and I1. There is only single fastq for each sample (5 samples = 5 FASTQ)

I expect that these are already merged. But, not sure. In this case, how to do the naming convention and how to check whether these are merged (R1, R2 and I1). Please suggest.

Thanks

[GenoMax]

Original format data appears to be available for these samples (R1 and R2). Here is one example: https://trace.ncbi.nlm.nih.gov/Traces/index.html?view=run_browser&acc=SRR12345675&display=data-access It may not be freely available though from cloud.

How did you download your files? Did you try the --split-files option to see if you can recover the two read files?

[shivangi.agarwal800]

Hi GenoMax,

I tried directly downloading from https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR12345675/SRR12345675 given on "Data access" tab and it downloads single file. I could not get option to download R1, R2 and I1. Also, I tried to grep "R2" from the downloaded file, but could not find any. Then, I tried following commands:

fastq-dump SRR12345675

fastq-dump SRR12345675 --split-3

The second command split SRR12345675 into SRR12345675_1.fastq and SRR12345675_2.fastq. Are these original read 1 and read 2 files ? And what about I1 index file ?

Please suggest.

Thanks

[GenoMax]

Hopefully those are the right files. You don't need I1 file since these samples are already demultiplexed. You will need to rename the files as SRR12345675_S1_L001_R1_001.fastq and SRR12345675_S1_L001_R2_001.fastq.