TABLE 1.

Pros and Cons when using multiplex PCR and 5′ RACE for TCR library construction

Items	Multiplex PCR	5′ RACE
Core principles	A series of mixed primers which are located on the known V allele are designed and paired with primers located in the J or C region.	This method relies on reverse transcriptase activity. Next, dCTP is added to the cDNA 3′ end as a primer binding domain during the first strand synthesis reaction.
Advantages	The method is compatible with gDNA and RNA.	(1) Can cover the entire V gene and retain the whole TCR and V/D/J region. (2) Avoid amplification bias.
Disadvantages	(1) Unable to detect mutation information for the V region. (2) Primer amplification bias	This method can only start with RNA, so it is more demanding than other techniques. Besides, the repeatability may be affected.
Correction	Adjust primer concentration or use a unique molecular identifier	‐

Abbreviations: TCR: T‐cell receptor(s); PCR:Polymerase chain reaction; 5′ RACE: 5′ Rapid Amplification of cDNA Ends