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Researchers Demonstrate Ion Torrent-based Sequencing Methods for B-cell Antibody Profiling

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NEW YORK (GenomeWeb) – Investigators at the Scripps Research Institute, the International AIDS Vaccine Initiative, and the Ragon Institute of Massachusetts General Hospital have come up with a set of next-generation sequencing techniques for profiling antibody repertoires of immune B cells — collections that provide clues for treating and tracking some forms of infectious disease.

"The idea is to capture the entire [antibody] repertoire, the entire population," Jiang Zhu, an immunology and microbial science researcher at the Scripps Research Institute, told In Sequence.

"It's like taking a snapshot of the immune system," he said. "You can use this to monitor immune response during infection … and also in clinical trials."

In a paper appearing in Scientific Reports last month, Zhu and his colleagues outlined their antibody profiling approaches, including an unbiased approach that uses 5'-RACE PCR and the Life Technologies Ion Torrent PGM instrument to sequence antibody variable domains of B cells — sequences too long to generate with short-read instruments and too diverse to tackle with lower-throughput technologies.

In parallel, the team demonstrated sequencing schemes that use single-molecule barcoding to more fully characterizing members of a given antibody family.

"We expect that, based upon long-read and high-fidelity next-generation sequencing technologies, the unbiased analysis will provide a more accurate view of the overall antibody repertoire while the barcoding strategy will facilitate high-resolution analysis of individual antibody families," authors of the study wrote.

For their proof-of-principle study, the researchers applied these approaches to samples from a well-studied individual with HIV-1, who was known to have developed so-called "broadly neutralizing antibodies." They also tested B cells from two individuals not infected with HIV-1.

In this and other studies, the group has shown that it's possible to produce functional antibodies based on the B-cell antibody repertoire sequences identified in individuals with HIV.

Because such analyses sometimes uncover antibodies more potent than those found using other approaches, Zhu argued that high-throughput B-cell antibody repertoire profiling could prove useful for discovering new antibodies for therapeutic use. Antibody profiles are also expected to prove useful for vaccine development and/or monitoring patients after vaccination or treatment.

Through somatic mutation and recombination of variable immunoglobulin gene regions, B cells acquire the ability to produce antibodies targeting a variety of infectious interlopers.

This mutation-prone B-cell maturation process leaves a genetic record of the challenges that the humoral arm of an individual's immune system has faced, Zhu and his colleagues explained.

While next-generation sequencing is an emerging area for assessing B-cell antibody repertoires, the methods have proven somewhat difficult due to biases associated with PCR methods used to amplify antibody sequences prior to sequencing, researchers explained.

Complications have also arisen when attempting to sequence antibody variable regions of B cells with technologies that offer inadequate read lengths, low throughput, and/or sequencing errors that obscure subtle antibody sequence differences.

"[A]ntibody repertoire analysis faces unique challenges in both sequencing and data analysis due to the complexity of B-cell development, in which antigen-driven affinity maturation selects for somatic mutations through variable regions of immunoglobulin genes," authors of the new study wrote.

"It is therefore critical to sequence entire antibody variable domains [about 450 base pairs] for a meaningful repertoire analysis and to recover functional antibodies from the [next-generation sequencing] data," they added.

While long read technologies are not critical for applications that include a relatively stable template sequence, Zhu explained, read length becomes crucial when considering antibodies, which are formed through sequence shuffling.

"You have to cover the whole gene," he said. "Otherwise, if you only sequence fragments, there's no way to assemble them back to the region where they originated."

Following the 5'-RACE PCR step, Zhu explained, the targeted sequence typically stretches some 600 base pairs in human samples. Some teams have turned to Roche 454 instruments to generate sequences long enough to span this region, he noted, though the cost and throughput has hindered more widespread use of that approach.

In an effort to curb the cost for such experiments and bump up the throughput, Zhu and his team decided to field test the Ion Torrent PGM platform, a technology with read lengths sufficient for seeing B-cell variable sequences and sequences introduced during sample preparation.

For their first attempt at sequencing full-length antibody variable sequences from B cells, the researchers focused on samples from an HIV-1-infected individual known as IAVI donor 17. Like some five percent to 10 percent of those infected with HIV-1, IAVI donor 17 had developed broadly neutralizing antibodies profiled in past studies, including a potent class of anti-HIV antibodies known as "PGT121" antibodies.

For their new study, the researchers started by using reverse transcription to convert immunoglobulin heavy- and light-chain antibody transcripts from the IAVI donor 17 individual's memory and plasma B cells from RNA to complementary DNA sequences.

From there, they PCR amplified and deep sequenced these regions with the Ion Torrent PGM, using the company's 316 chip and a sample preparation approach that skipped 3'-end trimming to maintain reads that were as long as possible.

By comparing the heavy- and light-chain sequences in PGT121 class antibodies to those found in the germline cells that produce such antibodies, the team showed that it could identify somatic mutations in the antibody variable region of the PGT121 antibodies that corresponded with those described by 454 sequencing.

Within the IAVI donor 17, the PGM-based analyses uncovered more than 1,000 PGT121 class antibody heavy chains and more than twice as many related light chains through deep sequencing experiments and an iterative phylogenetic analysis.

When they synthesized and tested a subset of those antibodies in the lab, the researchers found that they could reproduce anti-HIV-1 activity in a series of neutralization assays, consistent with the notion that the sequencing method was picking up functionally important alterations in the variable region of the PGT121 antibodies.

In subsequent stages of the study, the team found that incorporating random barcode sequences into the mix during the RNA-to-DNA conversion step could dial down sequence errors as well as noise associated with DNA amplification, leading to a more precise picture of a given antibody family.

To scrutinize a broader antibody repertoire in B cells from individuals with or without HIV-1, the team folded in a 5'-RACE PCR step prior to immunoglobulin heavy- and light-chain sequencing, using primers that matched a stretch of sequence downstream of the B-cell antibody variable domain region.

In samples from the HIV-1-infected individual and two HIV-free individuals, the researchers found that the 5'-RACE PCR and PGM sequencing method unearthed somatic variants in the heavy- and light-chain sequences of PGT121 antibodies in a manner that appeared to produce fewer biases than experiments done using multiplexed PCR.

The study's authors saw further boosts in PGM read length, throughput, and read quality — including a decrease in homopolymer errors — when they did similar antibody variable domain sequencing experiments with Ion Torrent's Hi-Q sequencing enzyme chemistry and an isothermal amplification approach that the company made available to them through an early access program.

"When we combined these two, we could easily get 600 base pair reads that were high quality," Zhu said. "The combination of IA and Hi-Q, plus some of our bioinformatics tools, gave us half of the repertoire without any gaps or insertion errors."

Even so, Zhu noted that the team is also interested in trying out Illumina sequencing instruments for antibody sequencing and has started discussions with that company to determine the most appropriate instrument and approaches.

Though the price of the approach changes with time as new reagents and chips are introduced, Zhu noted that the PGM-based approach currently costs a fraction of the price of NGS-based antibody experiments done in the past.

He and his colleagues are now applying the B-cell antibody sequencing methods for antibody discovery and treatment monitoring.

In collaboration with other research teams, for instance, they are using the method to track immune responses in individuals participating in clinical trials of vaccines for HIV, smallpox, yellow fever, hepatitis E, and human papillomavirus.

Zhu is also enthusiastic about extending the approaches to search for new antibodies in samples from individuals infected with other agents such as the Ebola virus.

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