QuestionsCategory: Single-Cell RNA-SeqDifference between 3′ and 5′ gene expression profiling? Which should I use? asked 2 years ago

For those looking to utilize high-throughput gene expression profiling methods that employ a transcript ‘end-counting’ strategy, one choice that has to be made is whether to employ a 3′ chemistry or a 5′ chemistry. Knowing when these are typically employed and what limitations each may have are an important early step in planning a single cell RNA-Seq study.
What is the difference between 3′ and 5′ gene expression profiling? Which should be used in which scenario?

2 Answers answered 2 years ago

For high-throughput droplet based gene expression profiling, 3′ came first and you will see many earlier studies use this format of chemistry. The 5′ method existed, but found wider adoption when it was adapted to droplet-based methods, such as on 10x Genomics Chromium. The major advantage and use of the 5′ was to allow both gene expression profiling and the identification of the TCR and BCR sequences on the same cells. As the version of chemistries improved, there was generally a higher sensitivity expected from the 3′ chemistry over the 5′ chemistry.  As newer version of 5′ chemistry is becoming available we will have to see how they compare in the near future in terms of sensitivity. Some groups that we’ve run 3′ and 5′ chemistry side-by-side for have found that even though they have higher gene detection rates on 3′ chemistry, they are okay with or in some cases prefer the data from the 5′ chemistry. One thing to keep in mind in this decision is that if you are using any “Feature Barcode” technology to make sure that it is compatible with the chemistry being used. For example, CITE-Seq BioLegend TotalSeqC products should be used with 5′ chemistry, whereas TotalSeqA would need to be used with 3′ chemistry.