Ultra-sensitive detection of rare T cell clones

Abstract

Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. The precision of the assay is demonstrated across five orders of magnitude. The accuracy for each clone is within an overall factor of three across the 100,000 fold dynamic range. Additionally, the assay is shown to be highly repeatable.

Figure 1. Expected versus observed frequencies for 6 spiked-in clones in multiple mixes

Four CD4+ clones (A, B, C, D) and two CD8+ clones (E & F) and were spiked into either complex PBMC or sorted CD4+ T cell populations at concentrations spanning five orders of magnitude. Results show concordance between expected and observed clone frequencies.

Figure 2. Experimental design and results of repeatability experiment

A) Schematic of experimental design. B) Sequence copy counts of TCRB clones obtained by sequencing the same PCR library on two different lanes of a flow cell. Each point represents a unique clone in this log-log scatter plot. The points in red are found in both repetitions, while those in blue where only found in the one lane. The total number of overlapping clones plotted on this graph is 122,000, with 20,000 clones sequenced that were unique to each lane. C) Sequence copy counts obtained by sequencing two separate PCR reactions performed using template from the same DNA extraction. D) Sequence copy counts obtained by sequencing two different DNA extractions from the same blood draw.