Rep-Seq: uncovering the immunological repertoire through next-generation sequencing

Abstract

Recent scientific discoveries fuelled by the application of next-generation DNA and RNA sequencing technologies highlight the striking impact of these platforms in characterizing multiple aspects in genomics research. This technology has been used in the study of the B-cell and T-cell receptor repertoire. The novelty of immunosequencing comes from the recent rapid development of techniques and the exponential reduction in cost of sequencing. Here, we describe some of the technologies, which we collectively refer to as Rep-Seq (repertoire sequencing), to portray achievements in the field and to present the essential and inseparable role of next-generation sequencing to the understanding of entities in immune response. The large Rep-Seq data sets that should be available in the near future call for new computational algorithms to segue the transition from 'classic' molecular-based analysis to system-wide analysis. The combination of new algorithms with high-throughput data will form the basis for possible new clinical implications in personalized medicine and deeper understanding of immune behaviour and immune response.

Figure 1

An overview of the process of obtaining the immunological repertoire. Starting with the relevant biological sample, T or B cells are isolated. According to the specific method used, recombined sequence regions are isolated and sequenced on a massive parallel sequencing machine. According to output sequences, clones are quantified.

Figure 2

Different PCR amplification techniques. (a) Multiple primers – two primers are designed to complement regions within the V and J segments. (b) 5′ RACE – only one primer is designed to complement the constant region of the cDNA. After the first amplification round, a homopolymer is synthetically added to the 3′. The cDNA is again amplified with the first specific primer and another primer targeting the homopolymer.

Figure 3

An account of the scales in the immunological repertoire, depicted through scales of water bodies. For example, sampling of the repertoire in human, is like samples a swimming pool using a single tablespoon. In zebrafish, the equivalent would be a 1:1 sampling. That is, current machinery enables the sampling of all the cells in the fish, or, in its scale parallel, sampling all of the water in a bathtub, utilizing all the water in the bathtub.