The human immune repertoire has a remarkable complexity of over 10^18 different T-cell receptors, and an even more diverse B-cell population. Recent interest in the Ion PGM System for immune repertoire sequencing has grown due to the extension of read-length to 400 bases, and the first publications are starting to appear.
Dr. Jiang Zhu (The Scripps Research Institute, La Jolla Calfornia USA) and colleagues recently published a paper studying the antibody repertoire of anti-HIV antibodies, and included several methods of reducing possible bias in the library preparation step. In collaboration with the International AIDS Vaccine Initiative (IAVI, New York New York USA), they focused their work on a sample known as IAVI donor 17, where they isolated B-cell RNA transcripts and compared the cDNA sequence to B-cell isolates from two HIV-1-uninfected samples.
Samples from IAVI donor 17 are of interest due to the presence of a powerful class of known anti-HIV antibodies that are broadly neutralizing, and are being studied for their potential use as an anti-HIV vaccine in the future.
By applying single-primer 5’-RACE (Rapid Amplification of cDNA Ends; the authors used Thermo Fisher Scientific’s FirstChoice® RLM-RACE kit) and turning off 3’ trimming in the Torrent Suite software (to get >500bp reads), the researchers were able to demonstrate less biased repertoires in comparison to gene-specific primers. They were also able to look at somatic variation in the repertoire sequences during the process of B-cell maturation, and from functional data demonstrated that the ‘PGM can be used to derive functional antibodies with a similar success rate (~70%) to 454’ method of sequencing.
By using random 10-mers and a single-molecule random barcoding scheme during the RNA to cDNA reverse transcription, they reduced PCR-based amplification artifacts, yielding better antibody-family phylogenic relationships.
They also were able to show improvements by an early-access use of both an improved Ion Sphere™ Particle template preparation kit, as well as the new Hi-Q™ Enzyme (previously described here in a video interview). To quote from the paper, ‘The combined use of the improved emulsion-based method and Hi-Q enzyme (new OT2 + Hi-Q) showed a remarkable improvement consisting of a 16% increase in the number of raw reads, a 50-60bp increase in read length…’ (p.9) The Hi-Q kit for the Ion PGM System is available now, while the new Ion OneTouch 2 Template Preparation kit* with the improved reagents will be available in the first half of 2015.
Dr. Zhu recently presented this work at the Genetic Solutions Tour in New York City, and you can access his video interview here.
Reference: He L, Sok D, Azadnia P, Hsueh J, Landais E, Simek M, Koff WC, Poignard P, Burton DR and Zhu J. “Toward a more accurate view of human B-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding”, Scientific Reports 4:6778 (2014) doi:10.1038/srep06778
*The content provided herein may relate to products that have not been officially released and is subject to change without notice