getting more estimated number of cells

[archana433]

I am doing cellranger count with my one sample, and we have used same GEW well and did resequencing of the same library to increase read depth. When I am combining the fastqc files and did cell ranger counts , I am getting estimated number of cells more than sum of the both runs.

I have used this command- cellranger count --id=sample_485_merged \ --fastqs=fastq_path/AAC7GKFHV,fastq_path/AAC2WMNHV/ \ --transcriptome=/scratch/tmp/vermaa/hg38/ \ --sample=sample_485,sample_485 \ --include-introns=false

1st run Estimated Number of Cells - 8,748 2nd run Estimated Number of Cells - 3,340 merged run Estimated Number of Cells - 12,260

Is my command is right ? Why I am not getting overlap from the same GEM well and same library?

[evolvedmicrobe]

In general, cell calling varies with sequencing depth so you shouldn't expect the same counts with both, you can learn more about our cell calling algorithm here

[archana433]

I will reframe this question i am trying 2 merge fastq files from the same sample but different sequencing runs. I now have two FASTQs files- one from the original shallow sequence and another from the new run. when I did cell ranger count , I am getting weird result. 1st run Estimated Number of Cells - 8,748. 2nd run Estimated Number of Cells - 3,340. merged run Estimated Number of Cells - 12,260

I am getting more cells, but there should be overlap because they are all from same GEM well.

cellranger count --id=sample_485_merged \ --fastqs=old_run/fastq_path/AAC7GKFHV,new_run/fastq_path/AAC2WMNHV/ \ --transcriptome=/scratch/tmp/vermaa/hg38/ \ --sample=sample_485,sample_485 \ --include-introns=false

Is my code correct? And how to resolve this problem? Why I am getting 12,260 cells while in my shallow seq it is 3,340 and another seq run - 8,748. I wouls have expecting more or less 8000-9000 cells not 12,260.

[elehnert10x]

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