Issue with FASTQs of 10X Flex with Antibody Capture
Hi,
I'm trying to analyze 10X Flex Data with Total-Seq C Antibody Capture, and I'm quite confused about the data I have at hand. For instance, my antibody hashtag sequences not only occur in Read2 of the AB FASTQs, but also in the gene expression FASTQs in Read2 (after the 10th base).
From my understanding, the AB hashtag sequences should only occur in Read2 of the AB FASTQs, is this correct?
Cellranger additionally complains that the barcode overlap between the AB FASTQs and the gene expression FASTQs is too low.
How could I debug these issue? I'm not that experienced with scRNA seq data. Thanks!
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The AB barcodes should be in the 10th base of the ADT library Read 2. Are you using
cellranger multifor quantification?Ensure there is no mix-up of FQ files.
Yes, I was planning to run
cellranger multi, and then further demultiplex withhashsolo.A low barcode overlap between the AB and gene expression would suggest that I have completely different cells, no?
Before speculating, lets get the facts straight: You got the fastq files with CellRanger
mkfastqand then proceeded withmulti, precisely following the manual?No, I got the FASTQ files directly from the sequencing facility, with
I1,I2,R1, andR2reads. Then I tried to run CellRanger, but it fails due to low barcode overlap.Then ask them whether they used mkfastq. It is what CellRanger expects for multi. CellRanger is highly custom and therefore complex, the exact expected input format must be provided.