Abstract:
:Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.
journal_name
Sci Advjournal_title
Science advancesauthors
Tanno H,McDaniel JR,Stevens CA,Voss WN,Li J,Durrett R,Lee J,Gollihar J,Tanno Y,Delidakis G,Pothukuchy A,Ellefson JW,Goronzy JJ,Maynard JA,Ellington AD,Ippolito GC,Georgiou Gdoi
10.1126/sciadv.aay9093subject
Has Abstractpub_date
2020-04-22 00:00:00pages
eaay9093issue
17issn
2375-2548pii
aay9093journal_volume
6pub_type
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