Help with error in cellranger

Question

[Chris]

I run this command:

cellranger-atac count --id=control1 --reference=/labs/reference_genome/Homo_sapiens_assembly38.fasta --fastqs=/labs/name_convention --sample=control1 --localcores=8 --localmem=64

[error] Your reference does not contain the expected files, or they are not readable. Please check your reference folder on smsh11dsu-srcf-d15-35.scg.

My fastq files are:

control1_S1_L001_R1_001.fastq.gz  
control2_S1_L001_R1_001.fastq.gz   
control1_S1_L001_R2_001.fastq.gz   
control2_S1_L001_R2_001.fastq.gz   

diseased1_S2_L001_R1_001.fastq.gz   
diseased2_S2_L001_R1_001.fastq.gz  
diseased1_S2_L001_R2_001.fastq.gz   
diseased2_S2_L001_R2_001.fastq.gz  

I added the index file .fai in the same folder with the reference genome. I am not sure the parameter for --sample in this case. Would you please have a suggestion? Thank you so much :)

[GenoMax]

[error] Your reference does not contain the expected files

The error is about the reference you are providing. Did you download pre-made cellranger-atac index files for human genome that 10x provides? If not that would be the best place to start. The index files are available on the same page where you download the cellranger-atac software.

[Chris]

I used other reference genome and index using samtools but not from their website. I will try that. Thank you :)

[Chris]

I found the reference genome but not the index, so should I download their reference and create the index file using samtools? Thank you so much.

[GenoMax]

That reference you see above contains all the files you need. Index, annotation etc. Download and extract. Then provide the path to the extract folder in your --reference option.

[Chris]

Thank you so much for the instruction : ) The past error has gone and a new error come.

Would you please have a suggestion? I only have R1 and R2 files but not I2.

Log message: Unable to read barcode sequence for read ID A01025:178:HTWY7DRX2:1:1101:19262:1031 1:N:0:TACTTGAA: there was no I2 read FASTQ and we were unable to read a 16 -base barcode from the FASTQ header. Make sure that the flow cell was demultiplexed correctly.

[GenoMax]

Can you show the output of: zcat control1_S1_L001_R1_001.fastq.gz | head -n 4?

Do your R1 files contain the necessary UMI+CellBarcodes?

[Chris]

This the output of your command:

@A01025:178:HTWY7DRX2:1:1101:19262:1031 1:N:0:TACTTGAA
CNATTCAAGGGTGGCTCTCCACTTCCGTTTTGGTTCAATGCC
+
:#FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:F:FFFFFF

I found this post has I1 files which I am not sure its role.

cellranger count error: No input FASTQs were found for the requested parameters.

[GenoMax]

How were these files created? Using cellranger mkfastq?

[Chris]

I am not sure. I was given these files with this explanation in the document:

Files with extension *.fastq.gz. Compressed files containing Illumina chastity-filtered reads with phred+33 quality scores.

[GenoMax]

Are you certain this is 10x single cell ATACseq data?

[Chris]

This is bulk ATAC seq data I have from a company name Active Motif. So cellranger is only used with single cell ATAC seq data from 10x genomics but not other companies?

[GenoMax]

cellranger-atac is for single cell 10x ATAC data. If you have bulk ATACseq data then you will need to use other programs.