Extended Data Fig. 1: Pooled TCR library subcloning and accuracy.
From: Discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening
(a, b) Schemes to produce ssDNA CDR3Jα pools (a) and ssDNA CDR3Jβ pools (b). Squares indicate 5′ phosphothioate modification. See Supplementary Table 29 for primer sequences. (c) Complete VCV pools can be PCR-amplified prior to subcloning using the common forward (CF) and reverse (CR) primers. Individual TCRs may be selectively amplified from VCV pools using the common forward primers (CF) and reverse primers targeting their respective Zip barcode sequences. (d) For the proof-of-concept TCR library (553 TCRs), the frequencies of individual CDR3Jα and CDR3Jβ sequences within the commercially synthesized CDR3Jα and CDR3Jβ ssDNA oligo pools and the assembled CDR3Jα-CDR3Jβ product were assessed by deep sequencing. The expected frequency of each CDR3Jα-CDR3Jβ pair was derived by multiplication of the observed frequencies of its respective CDR3Jα and CDR3Jβ sequences in the original, unassembled oligo pools. (e) Relation between Zip and CDR3Jα/CDR3Jβ oligo characteristics and the frequencies of resulting CDR3Jα-CDR3Jβ pairs after hybridization. (f) Relation between Zip and CDR3Jα/CDR3Jβ oligo characteristics and the accuracy of CDR3Jα-CDR3Jβ pairing (‘α-β pairing accuracy’).
