High throughput RNA sequencing

Once RNA is converted into cDNA it can be sequenced on an Illumina high throughput sequencer. Although on Oxford Nanopore type of devices, direct RNA sequencing is also possible.

Depending on the scientific hypothesis under investigation, the experimental design of an RNA sequencing experiment can be different. These parameters can be technical and/or statistical. These parameters will also have on effect on the total budget needed for the experiment. The main technical parameters to be determined are the following:

Type of library

• mRNA: only poly-adenylated RNA molecules are included in the library.
• small RNA library: only small RNA molecules are included in the library.
• ribosomal/mitochondrial RNA depleted RNA library (total RNA). All transcripts except ribosomal/mitochondrial and small RNA are included in the library.

Read size, single read or paired end reads

cDNA fragments can be sequenced to near completion or only partially. In the case of expression analysis, partially sequenced fragment will have enough specificity to be assigned uniquely to a gene and reflect the expression level of the gene.

If isoforms have to be quantified, sequencing the cDNA fragments to completion will generate more power to detect differentially expressed isoforms.

Sequencing coverage

Two factors will determine the numbers of reads per sample that have to be generated for an RNA sequencing experiment. The first one is the “size” of the transcriptome that one targets. The more transcripts are in the RNA mixture, the more coverage will be needed.

For instance sequencing an mRNA library will need less reads than sequencing a total RNA library, as the “space” one explore is bigger. A small RNA library will on the contrary be less demanding on number of reads than an mRNA library.

The second factor, is the expected expression levels of the genes of interest. Low expressed genes will demand a deeper sequencing in order to get an accurate quantification. An alternative approach for low expressed genes is an enrichment step, or by capturing the genes of interest, or by depleting highly expressed genes that are not of interest.

If you have an RNA sequencing experiment in mind come and discuss with us your experimental design.