GigaMune Rep-Seq

Technical Note

DEEP SEQUENCING OF THE T CELL REPERTOIRE Every person has more than 1 million unique T cell receptor (TCR) sequences generated by V(D)J recombination during T cell development (Warren et al 2011). These unique sequences define T cell antigen specificity. Deep sequencing of the T cell repertoire can measure repertoire diversity and identify T cell clones that have expanded in response to antigen stimulation. Capturing such high diversity requires multiplexed PCR amplification and library construction for next-generation sequencing, followed by extensive data processing. LIBRARY CONSTRUCTION
Genomic DNA

DATA PROCESSING STEPS Demultiplex samples Convert BCL to FASTQ format Identify V and J in each read Determine CDR3 sequence region Discard reads with uncalled bases or out-of-frame CDR3 regions Calculate clonotype frequencies for each unique CDR3 sequence

V
(59 genes)

D
(10e14 diversity)

J
(13 genes)

Genomic DNA

Forward primer set (20)

Reverse primer set (13) 1st PCR

NGS library adapter

NGS library adapter

Universal PCR Sequencing tag Sequencing tag

Warren RL, Freeman JD, Zeng T, Choe G, Munro S, Moore R, Webb JR, Holt RA (2011). Genome Res 21(5):790-7.

GigaMune Rep-Seq Technical Note 120914

GigaMune Rep-Seq is For Research Use Only. Not for use in diagnostic procedures.

Quality
1
Amplification bias. In any PCR-based technology, different primer sets may amplify with widely varying efficiency. A typical GigaMune Rep-Seq sample contains unique template DNA molecules spanning hundreds of possible V-J pairs. Because each of these pairs may amplify differently, accurate quantification of T cell repertoire data requires correction for amplification bias. We have constructed a positive control library that contains a defined mixture of known clones representing each possible V-J pair in a species. This library is amplified and sequenced in every sequencing run. From the abundance of each clone we calculate a correction factor for each V-J pair that we then use to adjust the abundances of clonotypes with matching V-J.

normalization threshold Abundance

All V-J pairs

Correction factor
2.0

1.0

0.5

Background subtraction. Every measurement technology detects some background noise, and T cell repertoire sequencing is no different. Detection and accurate measurement of a rare clonotype requires confidence that it arises from the biological sample. In GigaMune Rep-Seq, we distinguish background from signal by sequencing no-template negative controls to identify sequences due to primer-dimer, nonspecific amplification from genomic DNA, or other PCR artifacts. We then subtract this background from actual samples, leaving clonotype sequences most likely to have been generated by your experiment.

Reproducibility. There are several steps in the GigaMune Rep-Seq protocol at which sampling error might be expected to introduce wide variation in clonotype frequencies. We routinely run technical triplicates and produce pairwise whole-repertoire similarity matrices for an entire sequencing run to ensure high reproducibility.

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