CountPeaks about sierra

[rj-patrick]

Hi @AidenSb, yes that is correct.

Cheers,
Ralph

[rj-patrick]

Hi,

I don't think I've come across that error before, but the messages indicate a problem with the index file. Could you tell me if you regenerate the index (using samtools index) if that changes anything?

Cheers,

• Ralph

[JBreunig]

Thanks for the quick reply. I re-indexed the BAM file and got rid of that warning but still get the error:

There are 737280 whitelist barcodes.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
There are 64132 sites
Doing counting for each site...
Processing chr: 3
and strand 1
Processing chr: X
and strand 1
Processing chr: 16
and strand 1
Processing chr: 7
and strand 1
Processing chr: 11
and strand 1
Processing chr: 6
and strand 1
Processing chr: 13
and strand 1
Processing chr: 4
and strand 1
Processing chr: 9
and strand 1
Processing chr: Y
and strand 1
Processing chr: 17
and strand 1
Processing chr: 5
and strand 1
Processing chr: 2
and strand 1
Processing chr: 14
and strand 1
Processing chr: 10
and strand 1
Processing chr: 8
and strand 1
Processing chr: 15
and strand 1
Processing chr: 18
and strand 1
Processing chr: 1
and strand 1
Processing chr: 12
and strand 1
Processing chr: 19
and strand 1
Processing chr: MT
and strand 1
Error in { : task 1 failed - "1 elements in value to replace 0 elements"
In addition: Warning message:
In .get_cds_IDX(mcols0$type, mcols0$phase) :
The "phase" metadata column contains non-NA values for features of type
stop_codon. This information was ignored.

[rj-patrick]

Okay sure, I will download the bam file and see if I can replicate the error.

[rj-patrick]

Hi,

I was able to run CountPeaks on that data without any errors.

Can I check what reference you are using, i.e. is it the10x mm10 CellRanger reference? From your output, the error seems to occur while processing the MT chromosome - it is able to process other chromosomes? If there is a discrepancy in chromosome names that could become an issue.

[JBreunig]

Still getting this error even trying another E18 brain dataset (v2 chemistry).

Error in { : task 1 failed - "1 elements in value to replace 0 elements"

Can you just confirm the other input files in case I am doing something wrong in choosing inputs?
10X mm10 ref download: http://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-mm10-3.0.0.tar.gz
filename of ref from above link: mm10_genes.gtf
whitelist: 737K-august-2016.txt (from CellRanger 3.0 install)

[rj-patrick]

Hi,

The whitelist needs to be the list of cell barcodes from the experiment, so in the case of the mouse cortex data from above, download the 'Feature / cell matrix (filtered)', unzip the 'barcodes.tsv.gz' and provide the 'barcodes.tsv' file to CountPeaks. Hopefully that should fix the issue.

[JBreunig]

Ok, that did it...thanks!

[AidenSb]

@reprobate
Hi Ralph, I have a question about CountPeaks function.
from what I understood after we identify the peaks in the overall bam file, we count the UMIs that covering the peaks in each cell. we are basically counting the reads per peak and use that "read count" value as features (peak count) in a Seurat object for each cell, is this correct?
Thanks again for the responses.

Aiden