Roughly half the papers published so far for immune repertoire sequencing use genomic DNA samples, while the other half use RNA. Although we have primers that work for both sample types, our preferred originating template is RNA which we use to create an immune specific cDNA template.
The benefits using of RNA for immune sequencing are three-fold
First, using an RNA template allows us to maximize the amount of desired template we can add to the PCR amplification. Second, RNA is more sensitive than gDNA, because there are multiple copies per cell.
The main concern with using genomic DNA is inclusiveness. Each human cell may have about 6.6 pg of total genomic DNA of which the rearranged V(D)J represents a very small portion. So if 100ng of genomic DNA is used in the amplification, only about 16,000 cells are represented, which results in a highly restricted view of the repertoire. Although more genomic DNA could be used in the reaction, especially if concentrations are high, there is still a limit to the physical volume of DNA that can be added to the PCR chemistry. In contrast, the cDNA template created from iRepertoire’s RNA reagent systems will be immune-specific, maximizing the amount of desired template included in the reaction.
Finally, RNA filters out V- and J-gene segments that are not used in the recombination as only the expressed V(D)J segments are present.
Background noise is another concern with using gDNA as a template to generate a sequencing library. After V(D)J rearrangement, V and J segments not involved in the recombination will still be present in the genome and can serve as perfect binding sites for the primers. Binding at these sites will generate background amplifications, exhaust primers, and introduce bias.
Despite the downfalls mentioned, those who prefer to use gDNA as starting template have a variety of valid reasons. Samples are much easier to obtain, and even biopsied samples from tissue or slides can be used. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged VDJ will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. However, one can also argue that the RNA expression level of the T and B cell receptors may reflect the functional status better.
Importance of reverse transcription for immune repertoire sequencing
Customers can also opt to send cDNA created from oligo dT or random hexamer primers for downstream V(D)J analysis by iRepertoire. This may be useful if a single cDNA source was created for many different types of analyses, if you do not have the resources to ship RNA or other fragile materials, or perhaps if you have no remaining RNA but have cDNA template stored for the project of interest.
While we do accept cDNA samples, we recommend using our immune specific primers to create cDNA for several reasons. By using iRepertoire’s reagent systems for RT, the cDNA library will have an increased concentration of immune specific cDNA compared to a library produced from catch-all primers. The non-specific cDNA libraries include cDNA sequences from any expressed protein in the cell, which reduces the amount of immune specific cDNA template available for amplification. Furthermore, the creation of a robust cDNA library using non-specific primers requires a high RNA input.
iRepertoire’s amplification systems can reduce the amount of input RNA down to a recommended 100 ng (and we’ve had successful amplification with even less) because the cDNA library produced is immune specific.
So, RNA, gDNA, or cDNA? The answer really depends on your research goals and resources. If the repertoire changes you are looking for will be represented by dominant clones, then, either gDNA or RNA will work. If, however, you need to see the broader diversity or the true functional diversity, RNA is the better choice. If you need multiple types of sequencing analyses, pulling from one cDNA pool may be easiest and most cost efficient. Whatever your needs, iRepertoire has products and services to help reach your goals.