6.1.1.1. General Notes

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.