Question: Best way to perform single-cell RNA-seq normalization
0
gravatar for igor
3.0 years ago by
igor6.8k
United States
igor6.8k wrote:

Is there an optimal way to do single-cell RNA-seq counts normalization? Most RNA-seq normalization tools are designed with bulk samples in mind and assume a certain distribution of reads. With bulk RNA-seq, you have millions of reads per sample with relatively consistent coverage across samples. With single-cell experiments, you have much fewer total reads and many low expressing genes will be completely absent from many samples. I am curious how much I should worry about that and if there is a good way to account for that.

rna-seq single-cell • 2.3k views
ADD COMMENTlink modified 3.0 years ago by Devon Ryan85k • written 3.0 years ago by igor6.8k
0
gravatar for Devon Ryan
3.0 years ago by
Devon Ryan85k
Freiburg, Germany
Devon Ryan85k wrote:

For single-cell experiments people tend to normalize the spike-ins and use the resulting size factors. I would be very hesitant to use the actual gene/transcript/whatever counts for normalization in cases like.

ADD COMMENTlink written 3.0 years ago by Devon Ryan85k

Any particular tool we can implement the same? I understand DESeq uses the size actor for normalization. Does that mean DESeq is a good method to do it?

ADD REPLYlink written 3.0 years ago by Sandeep250
1

Yup, DESeq2 would work fine for this. There are some single-cell RNAseq oriented packages out there, such as SAMstrt. I don't work with single-cell data at the moment, so I can't say how good/bad things like SAMstrt are, but you should have a look at them.

ADD REPLYlink written 3.0 years ago by Devon Ryan85k

Will do. Thanks.

ADD REPLYlink written 3.0 years ago by Sandeep250

I am not sure about the spike-in controls. For example, Fluidigm (leading commercial solution) doesn't use spike-ins with the standard protocol.

ADD REPLYlink written 3.0 years ago by igor6.8k

Interesting, that makes life difficult. We use MARS-seq internally, cheaper and scales to many more cells.

ADD REPLYlink written 3.0 years ago by Devon Ryan85k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1852 users visited in the last hour