By Monica Heger
As next-generation sequencing gains more traction in labs, researchers are increasingly using the technology to understand more complex human systems such as the immune repertoire.
Because of its high variability, the immune system has been the subject of few sequencing efforts. But many researchers believe that sequencing the immune repertoire will provide important insights into the nature of disease and infection.
"This is a new field," Chris Carlson, a founder of Seattle-based Adaptive TCR, told In Sequence. "Assumptions we made 20 years ago [about the immune system] are not robust, and there are new and exciting things to be found using [sequencing] technology. I don't think we've even realized all the things we can do."
Carlson, who is also an assistant professor at the Fred Hutchinson Cancer Research Institute, was one of several researchers who discussed recent advances on immune repertoire sequencing during this year's Next Generation Sequencing and Genomic Applications Summit, held in Burlingame, Calif., earlier this month.
He said that sequencing the immune repertoire could have a number of important implications for understanding and treating disease. For instance, he said that sequencing T-cell receptors could help researchers understand autoimmune diseases like Type 1 diabetes or asthma.
These diseases are all associated with a particular HLA type, he said, but there are other unknown factors that determine whether a person will get the disease. Carlson said he thinks one possibility is that there is a particular T cell receptor that will initiate the disease in at-risk HLA types.
"If I can see your immune repertoire, then I have a snapshot of all the T cells in your system," he said. "If there were public T cells then I could look at a group of individuals that were affected and compare them to individuals with the same HLA type that were not affected. Then I could compare to try to find markers of the disease."
Carlson stressed that while the idea of a subset of T cells being shared among the human population is speculative, advances in technology would now make it possible to do investigate those types of questions.
Andrew Fire, a professor of pathology and genetics at Stanford University's School of Medicine, also predicted that sequencing would become an important tool for understanding immune response.
At the conference, he said that sequencing a person's immune repertoire could eventually help to monitor cancer by, for instance, enabling better initial detection, and by helping to determine whether cancer in remission has returned.
He cited a study he and his team are conducting in which immune cells from a patient with chronic lymphoma leukemia were compared to immune cells from a healthy patient. Early results of the study have detected clonal receptor sequences in the leukemia patient that were not detected in the control.
While the results have to be validated, Fire said that the goal is to identify sequences that are unique to the tumor that could be used as a marker to help diagnose the initial cancer and monitor its progression
Despite the potential of immune repertoire sequencing to yield important insights into the nature of infection, disease, and the body's immune response, it can be complicated due to the system's diversity.
As a result, companies like Carlson's and now Jian Han's iRepertoire, aim to reduce that complexity by providing sequencing services for customers.
"The immunorepertoire is the most dynamic portion of the genome," Han, also a faculty investigator at the Alabama-based HudsonAlpha Institute for Biotechnology, said. "It's at the front of the interaction between the environment and human genetics."
Han's company, a spinoff from the HudsonAlpha Institute, provides sample preparation, sequencing on Roche's 454 GS FLX, and data-analysis services, and sells the necessary reagents and instruments for automated library prep. He said iRepertoire will eventually offer sequencing on the Illumina Genome Analyzer.
iRepertoire made its debut in April with $2.7 million in initial funding from the HudsonAlpha Institute. Since then, the firm has raised an additional $4.3 million from private investors.
The key to sequencing the immune repertoire is in the sample-prep stage, Han said. But because the immune repertoire is so diverse it is difficult to prepare the library in an unbiased way. While a clinically relevant sample may contain as many as 20 million T cells and 5 million B cells, there may only be a few copies of a particular clone, so an amplification step must happen before sequencing. But, the amplification step must be sensitive enough to amplify all the targets, and in such a way that a particular target is not over or under-represented.
"If there's a minor difference in amplification efficiency, you will introduce so much bias in the 20 to 30 PCR cycles," he said. "And that is really the major challenge for immunorepertoire analysis."
His technology, amplicon-rescued multiplex PCR, or arm-PCR, is designed to thwart this problem. For each potential target, Han's team has designed two pairs of primers that are nested to each other.
The inside primers have a tag that can be recognized by a pair of communal primers. After the first round of amplification using the nested primers, the amplicons are recovered, and primers removed. Then, the communal primers are added for exponential phase amplification.
iRepertoire has also developed an instrument called iCubate to automate the entire process, Han said.
The company offers the library-preparation step as a stand-alone service, or in conjunction with sequencing and analysis. It will also barcode samples so up to 20 samples can be prepared individually and then pooled into a single sequencing run.
The library preparation costs about $1,000 per sample, he said, with 50 percent discounts for academic researchers and collaborations. Researchers can then either do their own sequencing and analysis, or iRepertoire can provide sequencing and analysis services.
Sequencing on the 454 platform will cost customers about $20,000; data analysis will add $10,000 per run.
Han said he had not yet determined the price for Illumina sequencing, but it would be less expensive than sequencing on the 454 platform. "It will be the more economical platform," he said. "But, we haven't fully validated it yet. Because it's a short-read platform, each read is a de novo assembly, so data analysis is really challenging."
In the future, Han said, the major market for iRepertoire and other companies in the field would be in clinical applications, which he said are a few years away.
Right now, customers of all the companies offering immune sequencing services will be "early adopters in this field, doing immunology analysis, vaccine studies, and looking for biomarkers for diseases," he said.