Ig-Seq/Rep-Seq/MAF
Ig-Seq/Rep-Seq/MAF
Rep-Seq is a collective term for repertoire sequencing technologies. DNA sequencing of immunoglobulin genes (Ig-seq) and molecular amplification fingerprinting (MAF)
In Ig-seq, is a targeted gDNA amplification method performed with primers complementary to the rearranged V-region gene (VDJ recombinant). Amplification of cDNA is then performed with the appropriate 5’ primers. Although throughput is high, in bulk analysis information regarding which VH and VL chains were paired in the same cell is lost, as cells are lysed in bulk and VH and VL genes are amplified in separate reactions. MAF (illustrated below) was developed to correct the amplification biases in Ig-seq, by using UIG tagging before and during the multiplex PCR amplification as well as a bioinformatics pipeline.
Pros:
- Measure antibody frequencies with up to 99% accuracy
Cons:
- The data analysis is challenging1
- In bulk analysis the information regarding which VH and VL chains were paired in the same cell is lost
- Ig-Seq: Vollmers C., Sit R. V., Weinstein J. A., Dekker C. L. and Quake S. R. (2013) Genetic measurement of memory B-cell recall using antibody repertoire sequencing. Proc Natl Acad Sci U S A 110: 13463-13468
- Rep-Seq: Benichou J., Ben-Hamo R., Louzoun Y. and Efroni S. (2012) Rep-Seq: uncovering the immunological repertoire through next-generation sequencing. Immunology 135: 183-191
- MAF: Khan T. A., Friedensohn S., Gorter de Vries A. R., Straszewski J., Ruscheweyh H. J., et al. (2016) Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting. Sci Adv 2: e1501371
- 1. Georgiou G., Ippolito G. C., Beausang J., Busse C. E., Wardemann H., et al. (2014) The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat Biotechnol 32: 158-168