Fig. 1

Pipeline overview and ST proportion distributions. (A) Overview of the TCR sequencing analysis pipeline: A. gDNA extracted from freshly resected murine peripheral blood, peritoneal orthotopic mesotheliomas, and spleen tissues. B. Equimolar mixture of synthetic TCR templates (ST) were then added, and C. followed by addition of forward and reverse sequences of ST (top) and TCRβ (bottom). For ST, universal 9-bp barcode (grey), unique 16-bp barcode (purple). For TCRβ, V-region (red), VD/DJ-junctions (grey), D-region (purple), J-region (green). D. Samples amplified with multiplex PCR followed by second-stage barcoding PCR. E. Samples were then pooled for sequencing. F. ST and TCRβ were then separated using universal barcodes, and G. ST was quantified using unique barcodes. H. TCRβ clonotypes were quantified with the MiXCR tool suite. I. Negative binomial normalization was used to remove amplification bias. J. Scaling factors were applied to counts, and K. then used to normalize counts for diversity analyses. Figure created with BioRender.com. (B) Plot showing stability for relative frequencies of ST counts within individual samples, and amplification bias based on the reproducibility of the multiplex PCR. Median IQR of ST-to-ST variation was ~20-fold greater than the median IQR of experiment-to-experiment relative frequencies of ST counts. Target values aim to be at the ln (1/260) line in the absence of amplification bias. Data derived from twenty ST-only samples described in the ST-only data sets