5.3. General Notes

AIR profiling of TCR and/or BCR chains:

• We recommend performing AIR DNA profiling separately for TCR (TRA, TRB, TRD, and TRG) and BCR (IGH, IGK, and IGL) chains considering significant differences in the representation of T and B cells which are present in many biological samples. E.g., In whole blood and PBMC samples, the number of T cells is 3-5-fold more than B cells. After the amplification step, which usually requires different cycle numbers for TCR and BCR chains, the amplified indexed products could be mixed together at equal amount for NGS step.

Starting Amount of DNA:

• For quantitative AIR repertoire profiling, we recommend using 2 µg of DNA (from whole blood, PBMC, lymphoid tissues, or isolated immune cells). Using larger amounts of DNA, e.g., by running technical replicates (see below), can improve the detection of rare clonotypes but requires increasing the sequencing depth (and NGS cost). Using smaller amounts of DNA will reduce the number of clonotypes that can be reliably profiled. For example, running 0.5 µg of total whole blood/PBMC DNA instead of 2 µg will profile ~100-200 highly abundant clonotypes (i.e., clonotypes with more than 10 target CDR3 DNA molecules) as opposed to ~500 clonotypes.

• Some samples such as FFPE blocks, or tumor biopsies contain impurities that inhibit the activity of DNA polymerase. To determine the optimal input amount for your samples, we recommend testing amplification efficiency using different starting amounts of DNA, e.g. 0.5 µg, 1 µg, 2 µg, 5 µg. Alternatively, to get better quantitative data, we recommend running samples in triplicates, e.g. using 2×3=6 µg of DNA for each sample as mentioned below.

• To achieve the most comprehensive AIR repertoire profiling for tissue samples with low content of immune cells (e.g., cancer biopsies, non-lymphoid tissues), it is recommended to purify lymphocyte cells (e.g., by FACS or antibody-magnetic beads) before DNA isolation.

Replicates:

• For samples with enough DNA, we highly recommend running technical replicates. We recommend increasing the reaction volume and amount of DNA at primer extension steps (e.g., 3-fold) splitting the samples after the Forward Primer extension step, and running the first and second amplification steps in triplicate reactions for each sample. Using technical or amplification triplicates allows estimation of variability and expansion confident quantitation from ~100 clonotypes if a single replicate is used to ~500 clonotypes based on the statistical power gained with triplicate analyses (see also the AIR Technical Guide for more details).

• For purified immune cell samples, we do not recommend splitting the samples into triplicates since the number of cells is usually limited. In such cases, we recommend collecting multiple samples for each cell fraction to run the biological replicates in parallel.

Positive Control DNA isolated from a single PBMC donor is supplied with the kit. We recommend running at least one positive control sample in a similar amount to your experimental samples. This will help in troubleshooting, data analysis, and normalization across multiple sample runs.