Methods for determining recombination diversity at a genomic locus
Inventors
Jayaprakash, Anitha Devi • Chess, Andrew • Sachidanandam, Ravi
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Assignees
Member
Icahn School of Medicine at Mount SinaiThe Icahn School of Medicine at Mount Sinai, located in New York City, is an international leader in biomedical education, research, and patient care. As the academic partner of the Mount Sinai Health System, the school is renowned for its innovative education, groundbreaking research, and commitment to health equity. With over 7,000 faculty, 1,200 students, and 2,500 residents and fellows, the institution fosters a culture of bold thinking, multidisciplinary teamwork, and a willingness to challenge conventional wisdom. Its mission is to radically advance the art and science of medical care through collaborative learning, scholarly inquiry, and a deep respect for diversity, preparing the next generation of healthcare leaders to revolutionize medicine and biomedical science.
The Icahn School of Medicine at Mount Sinai, located in New York City, is an international leader in biomedical education, research, and patient care. As the academic partner of the Mount Sinai Health System, the school is renowned for its innovative education, groundbreaking research, and commitment to health equity. With over 7,000 faculty, 1,200 students, and 2,500 residents and fellows, the institution fosters a culture of bold thinking, multidisciplinary teamwork, and a willingness to challenge conventional wisdom. Its mission is to radically advance the art and science of medical care through collaborative learning, scholarly inquiry, and a deep respect for diversity, preparing the next generation of healthcare leaders to revolutionize medicine and biomedical science.
Publication Number
US-12077753-B2
Publication Date
2024-09-03
Expiration Date
2035-11-20
Abstract
The present disclosure relates to methods for determining recombination diversity at a genomic locus of interest. The method includes fragmenting nucleic acids isolated from immune cells, ligating adaptors to the fragmented or amplified nucleic acids, and selectively amplifying nucleic acids containing a recombined junction at the genomic locus of interest. Selective amplification is achieved by using a first primer that hybridizes to an adaptor sequence and a second primer that hybridizes at a constant region downstream of the recombined junction. The selectively amplified nucleic acids may be sequences and analyzed to determine recombination diversity at the genomic locus.
Core Innovation
The invention relates to methods for determining recombination diversity at a genomic locus of interest, specifically focusing on T-cell receptor repertoires (TCRR) generated from complex populations of cells. The disclosed method involves isolating nucleic acids, such as mRNA from immune cells, fragmenting these nucleic acids to obtain fragments of a defined mean size, ligating adaptor modules to the fragments, and selectively amplifying nucleic acids containing a recombined junction at the genomic locus of interest by PCR using primers targeting both the adaptor and a constant region downstream of the recombined junction.
The problem addressed is the challenge of accurately and efficiently profiling highly diverse T-cell receptor repertoires, which are generated by DNA recombination events leading to receptor diversity critical for immune function. Existing methods for monitoring TCR repertoires suffer from amplification bias due to multiple primers with varying efficiencies, low specificity and efficiency with techniques like RACE-PCR, and difficulty in discovery of novel elements or extensive mutations. Furthermore, DNA-based approaches fail to distinguish between functional and non-functional copies, and conventional methods may miss the full diversity or accurate quantitation of the repertoire.
The invention provides a novel sequencing strategy termed T-seq, which uses universal primers ligated to fragmented mRNA and nested PCR with primers from the constant C region and universal 5′ adapter. This unbiased approach efficiently targets the recombined junction (such as the CDR3 region), recovering greater than 90% CDR3 reads for TCR α and β repertoires. The methodology reduces amplification bias, allows discovery of novel gene segments, and lowers sequencing costs. The resulting data can be analyzed to produce detailed annotations, characterize clonality, and generate comprehensive TCR repertoires, enhancing applications in disease diagnosis, monitoring, and personalized medicine.
Claims Coverage
The patent contains one independent method claim directed to generating T-cell receptor repertoires (TCRR) from T-cell populations. The inventive features focus on sample preparation, adaptor ligation, selective amplification strategies, primer design, and sequencing approaches.
Fragmentation and adaptor ligation of mRNA fragments
Fragmenting isolated mRNA to obtain fragments with a mean length less than 600 bp and ligating at least a first adapter module to the cDNA derived from these fragments, wherein the adapter ligates to a defined end of the cDNA.
Selective amplification using nested PCR with primers targeting adaptor and constant region
Performing first and second rounds of PCR amplifications using primers where the first primer binds at least partially within the first adapter module and the second primer binds within the constant (C) region downstream of the recombined junction, with the second PCR using nested primers that bind at positions yielding a shorter nucleotide distance than the first PCR, enhancing specificity and covering the recombination junction (e.g., CDR3).
Primer design incorporating barcodes and hybridization regions
Use of primers where the constant region binding primer includes portions: a hybridization region, a barcode region, and a sequence that hybridizes specifically to the constant region downstream of the recombined junction, enabling multiplexed sequencing and sample tracking.
Use of indexing sequences in adaptor modules for clonal analysis
Adapter modules include indexing regions with sequences that allow distinguishing between different samples or clonal expansions by tracking adaptor-associated barcode sequences across recombined junction sequences.
Sequencing and annotation of amplified fragments
Sequencing the selectively amplified nucleic acids and annotating the recombined junction sequences to assemble a comprehensive recombination profile of the subject, and optionally comparing this profile to reference profiles characteristic of medical disorders.
The independent claim covers a method for efficiently generating accurate T-cell receptor repertoires by fragmentation of mRNA, universal adaptor ligation, and selective nested PCR amplification with cleverly designed primers targeting the adaptor and constant region to enrich recombined junction sequences, enabling sequencing, annotation, and clonality analysis with minimal bias and high coverage.
Stated Advantages
Overcomes limitations of existing TCR repertoire analysis methods, including PCR bias introduced by multiple primers.
Highly efficient recovery of recombined junction sequences (e.g., >90% CDR3 reads), enabling accurate and unbiased repertoire profiling.
Allows discovery and characterization of novel TCR gene segments and alleles not annotated in existing databases.
Reduces sequencing costs through targeted amplification and sequencing strategy.
Enables quantification of clonality and tracking of individual recombined sequences through use of molecular barcodes and indexing.
Improves the interpretation of TCR repertoires in clinical applications such as diagnosis, prognosis, and monitoring of autoimmune diseases like Graves' disease, infections, and other immune-related conditions.
Documented Applications
Profiling T-cell receptor repertoires from immune cell populations, including sorted T-cell subtypes such as CD4+, CD8+, Tregs, and memory T-cells.
Analyzing TCR repertoire diversity in the context of autoimmune diseases, exemplified by Graves' disease, for diagnosis, monitoring treatment progress, and identifying therapeutic targets.
Characterizing TCR repertoires in humans and mouse models for understanding immune responses and genetic influences such as HLA haplotypes on TCR diversity.
Studying clonality and repertoire diversity changes in infections, transplantation, allergy, and immunization/vaccine responses.
Profiling B cell receptor repertoires (BCR) to study B cell infiltrants in diseased tissues such as cancer and autoimmune thyroid tissue.
Developing biomarker signatures for immune-related diseases and enabling personalized medicine approaches based on detailed profiling of immune receptor repertoires.
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