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Low number of genes per cell but high sequencing saturation

Posted By: cap76, on Aug 28, 2018 at 10:17 AM

I'm trying to figure out possible sources of error in a recent 10x run and was wondering if anyone had encountered a similar situation.

 

The median number of genes per cell was very low (<100) and so was the mean reads per cell (well below the recommended 50k/cell) but Sequencing Saturation was high (>98%). Presumably this would suggest a limtited return from a greater sequencing depth. Has anyone else seen this before?

3 Replies

Re: Low number of genes per cell but high sequencing saturation

Posted By: dporter, on Aug 29, 2018 at 3:42 PM

My guess would be that you sequenced a lot of PCR products. What does your bioanalyzer trace look like? Do you see a large peak less ~100 bp? 

Re: Low number of genes per cell but high sequencing saturation

Posted By: Daha, on Sep 13, 2018 at 6:45 AM

We have experienced exactly the same problem in two recent experiments. The size distributions of cDNAs (Bioanalyzer traces) look good, but seqencing saturation is very high, even after shallow sequencing. Data quality is also very poor compared to previous runs.

 

Did you manage to solve your problem?

Re: Low number of genes per cell but high sequencing saturation

Posted By: cap76, on Sep 13, 2018 at 6:57 AM

Thanks both for the comments. I wasn't involved in the inital experiment, but it was done through a facility that has a reasonable amount of experience, so I'd imagine the Bioanalyzer traces would have been fine. Will take a look to double check.

 

I'm guessing this isn't merely a case of sequencing deeper i.e., we've already found most of the unique molecular species we're going to find in the population as a whole, and just need to sequence more to get an more accuarte count of those species in any given cell?

 

Will speak to some more people soon, and post if we get to the bottom of this.