Sample Type: RNA vs. gDNA
About half of 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 (see below), we prefer RNA as a starting template, if possible.
The main concern about using genomic DNA is the inclusiveness. There is only a certain amount of template DNA that one can add into a PCR reaction. Each human cell may have about 6.6 pg of genomic DNA of which the rearranged V(D)J represents a very small portion. So, if 100ng of genomic DNA is used in the reaction, this represents only about 16,000 cells, resulting in a restricted view of the repertoire. Although more genomic DNA can be used, there is a limit to how much can be added. 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, and excluding unnecessary and potentially disruptive gDNA.
A second concern about using genomic DNA as a template to generate a sequencing library is the background. As the figure below demonstrates, after the V(D)J rearrangement, those V and J segments not involved in the recombination will still be 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.
V(D)J rearrangement demonstrating the increased background that may occur due to the extraneous priming sites available within gDNA. This can generate background amplification, exhaust primers, and introduce bias.
Those who prefer to use genomic DNA 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. So, gDNA or RNA? The answer really depends on your research goals. If the repertoire changes you are looking for will be represented by dominant clones, then, both gDNA and RNA will work. If, however, you need to see the broader diversity, RNA may be better.
Sample Type: RNA vs. cDNA
What about using RNA directly versus a cDNA library as a template in the reaction? We recommend using our primers to create cDNA rather than using poly dT primers or random hexamer primers for several reasons. By creating the cDNA using iRepertoire’s reagent systems, the cDNA library will have an increased concentration of immune specific cDNA relative to a library produced from catch-all primers. These non-specific cDNA libraries will include cDNA sequences from any expressed protein in the cell, reducing the amount of immune specific cDNA template available for amplification. Furthermore, the creation of a robust cDNA library using non-specific primers requires a lot of input RNA. Wecan reduce the amount of input RNA down to a recommended 100 ng (however, less may also work) because the cDNA library produced will be immune specific.
Sample Type: Selecting the Correct Reagent System
The type of template you decide to work with will affect the type of primers you should use. All of our reagent systems work with RNA; however, due to an intron between J and C genes, only V-J primers should be used with genomic DNA. Currently, for the Illumina platforms (GA II, HiSeq2000, and MiSeq), we have both the V-J and V-C primer designs for the human TCR beta chain. The V-J primers amplify about 100bp sequences around the CDR3 region and are suitable for both genomic DNA and RNA samples; the V-C primers amplify about 150 bp sequences around the CDR3 region and are appropriate for RNA samples only due the previously discussed intron. For mouse TCR alpha and beta chains, we currently offer the V-C primer system (RNA only). Please contact us if there is a set of V-J primers for the Illumina platform that you would like to use not currently listed in our catalog.
For B cell studies, we have V-C gene primers only for both Illumina and Roche 454 systems. For those want to obtain antibody sequence information beginning in first framework region through a portion of the C-region, the Roche 454 platform or the Illumina MiSeq 250 PER is recommended as it will give read length of over 400bp.