Measuring and monitoring of cell clonality
Methods are provided for the detection and analysis of clonality in a cell population, where parallel sequencing is applied to a nucleic acid sample obtained from the cell population, optionally a population of lymphocytes. Replicate samples are amplified, and sequenced, where identification of coincident sequences in two or more replicates is indicative of clonal expansion.
1. A method of determining in an individual when a complex cell population contains a clonal expansion of a subset of cells that share a genetic sequence at a locus of interest, wherein the genetic sequence at the locus of interest is distinct from germline sequence of the individual, and wherein the cell population exhibits high sequence heterogeneity at the locus of interest, the method comprising:
dividing said complex cell population comprising said subset of cells that share a genetic sequence at a locus of interest, or nucleic acids derived therefrom, into at least two distinct pools;
amplifying nucleic acid sequences selected from DNA, mRNA or a mixture of DNA and mRNA obtained from said at least two distinct pools of said complex cell population, at the locus of interest;
sequencing at least 10 3 reads from the amplified nucleic acid populations at the locus of interest from said at least two distinct pools;
comparing sequences of said amplified nucleic acids at the locus of interest, to detect the presence of sequences that are coincident in said at least two distinct pools of nucleic acid;
wherein the presence of coincident sequences in said at least two distinct pools of nucleic acids at the locus of interest, having a sequence distinct from germline, is indicative that the complex cell population comprised a clonal expansion in a subset of cells that share a genetic sequence at a locus of interest.
2. The method of claim 1 , in which the sequences at the locus of interest are amplified using oligonucleotide primers with homology to specific regions of the genome.
3. The method of claim 2 in which the oligonucleotide primers are capable of selecting or amplifying segments of the genome encoding the immunoglobulin heavy chain, the immunoglobulin kappa light chain, the immunoglobulin lambda light chain, the T cell receptor alpha chain, the T cell receptor beta chain, the T cell receptor gamma chain, or the T cell receptor delta chain.
4. The method of claim 2 in which the method of amplification is a polymerase chain reaction, a rolling circle reaction, a ligation-mediated amplification, or a multiple displacement amplification.
5. The method of claim 1 , in which the individual is a non-human animal.
6. The method of claim 1 , in which the individual is a human.
7. The method of claim 1 , in which the individual is a patient with possible or confirmed neoplasm, possible or confirmed hematologic neoplasm, possible or confirmed autoimmune disorder, possible or confirmed allergic disorder, possible or confirmed atopic disorder, possible or confirmed immunodeficiency disorder, possible or confirmed infectious disease, possible or confirmed organ rejection, possible or confirmed transplant rejection, possible or confirmed transfusion reaction, possible or confirmed immune-mediated condition.