Cellranger count creating an empty matrix.mtx after adding 'N' and '#' to R1 UMI and quality respectively to account for 9bp UMI.

Hi!

I am currently analysing public scRNA-seq datasets from raw reads.

For a public dataset, the R1 read length is 25. The library was generated with an older version of the chemistry. This throws an error in cellranger as the read length is too short.

To handle this, 'N' were added to the end of the R1 reads as described in this 10XGenomics page via a simple sed command.

This paper describes adding 'A' to the UMI to handle this.

A subsequent error was raised with the message Sequence and quality length mismatch. This was dealt with by adding an '#' to the quality score line to coincide with the 'N' of the sequence.

A R1 read length of 26 was achieved.

Example:

Prior to UMI length adjustment:

@ST-K00127:336:HVYF2BBXX:1:1101:23744:1244 1:N:0:GAGNATNT
GGGCACTTCTTGNATTTCTGTTTTC
+
AAFFFJJJJJJJ#FFAJJFJAJJFJ

Post UMI length adjustment:

@ST-K00127:336:HVYF2BBXX:1:1101:23744:1244 1:N:0:GAGNATNT
GGGCACTTCTTGNATTTCTGTTTTCN
+
AAFFFJJJJJJJ#FFAJJFJAJJFJ#

Cellranger count ran and completed with a success message.

However, it was noticed that the filtered_features_bc_matrix matrix.mtx.gz and the raw_features_bc_matrix matrix.mtx.gz files are empty bar the header:

Example:

%%MatrixMarket matrix coordinate integer general
%metadata_json: {"software_version": "Cell Ranger 4", "format_version": 2}
36601 551223 0

I'm assuming this is due to the handling of the UMI. Is there a way to resolve this?

Thanks!

$ cellranger --version
cellranger cellranger-7.0.1

10XGenomics / cellranger

Cellranger count creating an empty matrix.mtx after adding 'N' and '#' to R1 UMI and quality respectively to account for 9bp UMI. #202