The immune repertoire reflects the sum total of diverse B and T-cells in the circulatory system. The adaptive immune system drives immune response by these hypervariable molecules. The antigen specificity of each T-cell receptor (TCR) is determined by the complementarity-determining region: CDR3 of the beta receptor chain, formed by V, D and J gene regions. Examination of TCR diversity is important for understanding adaptive immunity and it’s function in diseases. Next generation sequencing has become a powerful tool for measuring TCR diversity. Before samples can be sequenced a unique library preparation method must be performed to allow for reproducible and reliable results.
Girihlet, a newly formed biotech company in Brooklyn, NY is one of the first companies to offer TCR repertoire sequencing services and is the first to offer it on Genohub.com. We got in touch with Girihlet to learn more about this service offering and have posted our conversation with one of it’s co-founders, Dr. Ravi Sachidanandam. Ravi also holds a position as Assistant Professor on the faculty of the Icahn School of Medicine at Mount Sinai, department of Oncological Sciences. He has published over 85 papers in the latest and most interesting areas of genomics, including small RNA, mRNA splicing, methylation and virology.
Genohub: Hi Ravi, we’re excited that you’ve joined Genohub.com and listed your services. We’re particularly interested in the TCR-repertoire sequencing services you have on Genohub.com. Not many service providers currently offer this service, how come?
Ravi: There are very few companies that offer this currently, and this is mostly because it’s a very challenging problem both experimentally and computationally. It may be easier to count all the dollar bills in circulation than to profile the diversity of the T Cell Receptors.
Genohub: Can you comment briefly on the ‘library prep’ approach to TCR profiling?
Ravi: Our library prep method is very unique, it is based on quantifying RNA, and in particular just the CDR3 regions while most of the other companies quantify DNA. This allows us to only quantify functional rearranged TCR locus. We also use universal primers for amplification and do not depend on previously known TCR regions, thereby accelerating discovery. We have also compared our data to flow results and demonstrated good concordance.
Genohub: Inefficiencies during library prep and and sequencing can lead to severe bias generating artificial TCR diversity. Does your approach address this?
Ravi: The beauty of our approach is we use common primers to amplify the T cell receptor regions. This ensures there is no bias during PCR, allowing for accurate sequencing. And since the accuracy and enrichment for the TCR mRNAs is >98%, we need very little total RNA and less sequencing depth reducing the overall cost of sequencing.
Genohub: How many sequencing reads or TCR sequences do you recommend for a single human sample? Our readers can use your recommendation directly on our project search page: https://genohub.com/shop-by-next-gen-sequencing-project/.
Ravi: Currently 10 million sequences of 150bp PE reads is enough to accurately and quantitatively capture most of the TCR diversity
Genohub: How do you handle under-expression?
Ravi: We keep track of low -expressed TCR transcripts as they are needed to understand the statistics of the distribution of the TCR repertoire. We provide these to the researchers, in case they might need to look for rare transcripts.
Genohub: Why is diversity of the immune repertoire important for health?
Ravi: The diversity is the key to the effectiveness of the TCR-repertoire. The diversity reflects the ability of the immune system to fight infections.
Genohub: Any comments on its use for vaccine development, autoimmune study, biomarker detection?
Ravi: We believe the TCR sequence can be easily monitored over time, thereby serving as a powerful biomarker to study the effects of vaccination, to determine if the vaccination was effective. It will also be useful in understanding the underlying cause of autoimmune reactions.
Genohub: Thanks for taking the time to discuss this exciting new method. Is there anything that you’d like to add?
Ravi: Girihlet is very excited to take this approach to the rest of scientific community and make a significant difference on how the TCR is sequenced currently and eventually have an impact on the practice of “precision medicine”.