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Roche's 454 Eyes Immune Repertoire Sequencing as Key Application for Long-Read Platform

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By Monica Heger

Immunogenetics is likely
to be a key and growing application for 454 sequencing, Michael Egholm, vice president of research and development at Roche's 454 business told In Sequence last week.

While some researchers are looking at short-read platforms such as the Illumina Genome Analyzer for immunogenetics applications, Egholm said that the long, accurate read lengths that can be achieved using 454's technology make it ideal for immune profiling.

The relevant sequences on the immune cells — the areas that respond and bind to specific antigens — are around 100 bases long, plus researchers would also need to have the sequences on either side of that region. As a result, said Egholm, it is important to have accurate read lengths of several hundred base pairs in order to characterize the immune system and see how it reacts and responds to disease. He said that the long read lengths of 454 — currently in the range of around 400 or more base pairs — make it an ideal platform for immunogenetics research. "This is really in our sweet spot," he said.

Two recent proof-of-principle papers underscore potential applications of immune repertoire sequencing on the 454 platform, said Egholm. In a paper published in Science Translational Medicine last month, Andrew Fire's group from Stanford University sequenced the B cell population in a clinical sample of both healthy patients and cancer patients on 454's GS FLX. And, in a separate paper published in December in the Proceedings of the National Academy of Sciences, researchers from the HudsonAlpha Institute for Biotechnology sequenced human T-cells from one individual, also with the GS FLX.

The HudsonAlpha group used 454 to characterize the diversity of a T-cell repertoire from a healthy male. The team obtained read lengths of 250 base pairs and generated 1.67 million sequence reads for eight distinct T-cell populations. From the sequence reads, the team identified 1.48 million CDR3 intervals — the area that binds to the antigen.

Jian Han, senior author of the study, said the findings demonstrate just how complex one person's immune repertoire is. Eventually he thinks it will be possible to profile an individual's immune repertoire and identify previous infections.

Fire's group at Stanford sequenced the B cells of both healthy human samples and samples with hematological malignancies. His team designed amplicons with average lengths of around 350 base pairs using both the standard GS FLX, as well as the Titanium.

Unlike Han's group, which was trying to characterize variation within one healthy individual, the Stanford study was more of a survey looking for broad differences between a healthy and a cancer immune repertoire.

They were able to identify some expected differences. For example, in the cancer patients, they found a large number of B-cell clones, indicative of the immune system's attempt to produce useful antibodies to fight off the cancer.

Fire said that having long reads was critical for his work. There are "rearranged segments that have several hundred bases of important information. It's hard to imagine getting that without longer reads," he said. "One way or another, you're going to need extended information about individual molecules."

He said that one area of immunogenetics he is interested in is autoimmune disease, like multiple sclerosis, where the immune system responds inappropriately. "Are there any precipitating events that you can see — viral infections, bacteria infections, other events that could be there?" he said.

Aside from basic disease research, Egholm said 454's quick runtime could make it a particularly suitable diagnostic tool for patients with unknown or difficult to diagnose illnesses. He envisions sequencing a patient's immune repertoire while they are at the hospital.

"The vision we have is that one day this will be a standard test. When a patient walks in with a nondescript disease, along with other tests, we'll also sequence the immune system," Egholm said. "The ultimate vision is that we'll be able to translate the actual sequence of the VDJ combination back to what the patient was exposed to."

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Roche has previously said that another application of the 454 technology is in HLA typing, which is used in bone marrow transplants and matching tissue in organ and tissue donor surgeries (see In Sequence 3/17/2009).

Egholm added that the company continues to explore HLA typing, but is also moving into projects focused on the immune system's response to vaccines and viruses, and trying to characterize how the system begins to produce antibodies. While he declined to give specifics on the different research projects since they haven't yet published results, he said that there are groups working on HIV, as well as the hepatitis B and C viruses. Sequencing both the viruses themselves, as well as the immune cells after they've been exposed to viruses, could enable researchers to find new drug targets as well as where in the virus resistance is developing, he said.

Egholm thinks that eventually all the data could be combined to get a better understanding of the immune system and how it responds to disease. "If you take the sequencing of viruses and HLA, and now the immune cell sequencing, you're attacking the whole infection and immune response from different angles," he said.

Meanwhile, other researchers have used different approaches to solve the problem of sequencing the immune repertoire. Jay Shendure of the University of Washington recently developed an approach he dubbed subassembly (see In Sequence 1/19/2010), which uses tags to create longer fragments from short reads on the Illumina platform. He told In Sequence that one application he envisions for the approach is profiling the immune repertoire.

Also, Robert Holt, senior scientist and head of sequencing at the British Columbia Cancer Agency's Genome Sciences Centre, said that Illumina's read lengths, of just over 100 base pairs, are now reaching the point where they are viable for immune cell sequencing. Last year, for example, he and his colleagues published a study in which they profiled the repertoire of T-cell receptor beta chains using the Illumina Genome Analyzer (see In Sequence 6/30/2009).

Holt said that Illumina has an advantage in that it generates so much more data, so researchers are sequencing the same areas many times. This is particularly important for immune cell sequencing because the regions of interest are so variable that you would want to have sequenced them many times to ensure that any variants detected are actual variants and not sequencing errors, he said.

Illumina has also signaled that it aims to move in this direction itself. Greg Heath, senior VP and general manager of Illumina's diagnostics business unit, said during the firm's recent R&D day for investors and analysts that it plans to develop a sequencing-based HLA assay in its CLIA lab this year.

Holt said that both Fire's and Han's papers are good examples of what is starting to be done with immunogenetics, but that they both still had limitations. For example, he said Fire's technique is a useful approach for determining obvious differences between the immune repertoires of healthy patients versus cancer patients. But, he added, it is limited in that the researchers didn't sequence to a great enough depth to allow them to compare immune repertoires between healthy individuals. "They find very little overlap among the repertoires [of healthy individuals], but with that low coverage, if you sequence the same individual, you'll also find very little overlap. So, you're not capturing the full diversity," he said.

Understanding the full diversity among healthy immune repertoires will be a key to making immunogenetics truly useful, Holt said. Only then will it be possible to identify the relevant variants in aberrant immune repertoires. Nevertheless, he said immunogenetics is becoming an exciting new area, and these two studies demonstrate the potential of next-generation sequencing in immune profiling.

"Previously we've been almost completely blind to immune diversity," said Holt. "Now, we're at the point of being able to address that [diversity], and it will open up important new windows on exploring both the healthy and unhealthy immune system."

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