scRNA-Seq GEX 5' v1.0 High Cell Count with Low Gene Count
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3.2 years ago
tdfyoder ▴ 40

Hello,

My lab has launched our first 10x GEX 5' libraries (v1.0). Cell viability was good (96%) and cDNA/SI-PCR product traces matched QC. Our strategy was downsample the libraries after over-sequencing to determine optimal depth for the cell type ( and aid in detecting the transgene). BCL's were converted to fastq via CRv.5.0.1. A custom reference was generated using the same CR. Our web summary html from CR count showed we had greater than expected cells (23K; 17.5K loaded expecting 10K) and a low median gene count of 131 genes.

Prior to launching CR count, I downsampled the fastq's using seqkt sample. With matching random seeds for R1/R2. I am wondering if this has contributed to the skewed cell/gene count?

Previously, our lab has used CR 3 to analyze 5' v1.0 data.

Right now I am consider rerunning with CR3 and pushing the full fastq (800million reads) through CR and downsampling afterwards.

10x tech support suggested a cell prep/lib prep issue either poor quality cells, GEM emulsion failure or premature lysis.

We did not sequence libraries in channels 7 & 8.

GEMs post chromium rn

Any thoughts, advice would be appreciated. Thanks.

-Todd

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10x scRNA-seq downsampling • 1.1k views
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Entering edit mode
3.2 years ago
tdfyoder ▴ 40

It was determined this as a loss of single cell behavior due to a wetting failure (reagents comped).

If running cellranger count on hpc with '--nosecondary' option resulting web_summary.html will not include t-SNE plots/DEGs. These plots are necessary for troubleshooting.

wetting failure

tech note

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