Figure 2.

Library preparation strategies for antigen receptor HTS. The extraordinary diversity of antigen receptor sequences poses challenges for targeted amplification and library preparation; although not comprehensive, a general overview of select PCR priming strategies is presented here. A generic antigen receptor schematic representative of BCR heavy or TCRβ is shown. (A) PCR amplification and library preparation from genomic DNA. Multiplex PCR strategies, in which complex mixtures of primers complementary to many or all possible V segment sequences, can be used to amplify portions of variable region sequences from genomic DNA. Multiplex primers targeting portions of V segments (red) can be used in conjunction with J segment primers (navy blue) for amplification. Upon incorporation of HTS adaptors (light gray boxes), long read HTS can be used to capture a majority of variable region sequence. Alternatively, short read HTS can be used to sequence only the CDR3 region. (B) Reverse transcription PCR amplification and library preparation from mRNA. In antigen receptor transcript mRNA, the juxtaposition of constant region exons adjacent to the variable region offers a reverse priming site with minimal diversity. In invariant adaptor strategies, the complexities and potential biases of V segment multiplex PCR are bypassed by incorporating a defined adaptor sequence (dark gray box) upstream of the variable region by oligonucleotide ligation or template switch methods during reverse transcription. A single primer to the adaptor sequence (dark gray) can then be used with constant region primers (violet) to generate amplicons that contain complete variable region open reading frames (ORFs). These can be sequenced in entirety by long read paired-end HTS or sequenced with short read HTS for CDR3-targeted studies. Similar adaptor-mediated PCR strategies using multiplex J primers for CDR3 sequencing are also available (not shown). Alternatively, following reverse transcription without invariant adaptors, multiplex primers to V regions and J regions (as for genomic DNA amplification) or constant regions (not shown), can be used for CDR3-targeted short read HTS. Hashed lines, HTS reads. V, D, J, and constant segment colors as in Figure 1. Abbreviations: BCR, B cell receptor; TCR, T cell receptor; V, J, and D, Variable, Joining, and Diversity gene segments; HTS, high throughput sequencing; CDR3, complementarity determining region 3.