Quantification of adaptive immune cell genomes in a complex mixture of cells
Compositions and methods are described for highly sensitive quantification of the relative representation of DNA from adaptive immune cells (e.g., T and/or B lymphocytes) in DNA extracted from complex mixtures of cells that include cells which are not adaptive immune cells. Included are methods for determining the relative presence in a tumor of tumor infiltrating lymphocytes (TIL), the relative presence of lymphocytes infiltrating a somatic tissue that is the target of an autoimmune disease, and the relative presence of lymphocytes infiltrating a transplanted organ.
1. A method for assessing the prognosis of a subject receiving therapeutic treatment for a solid tumor based on the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in said solid tumor comprising:
a) obtaining a first sample from a solid tumor from a subject at a time point prior to therapeutic treatment and a second sample from the solid tumor from a subject at a time point after to therapeutic treatment;
b) extracting DNA from said first and second samples;
c) amplifying in a multiplex PCR reaction substantially all rearranged TCR or Ig CDR3 encoding regions present in said sample samples using a plurality of V segment oligonucleotide primers comprising sequences selected from the group consisting of SEQ ID NOS: 1-52, 221-238, 255-260, 262-267, 269, 272, 283, 286, 291, 292, 294-297, 301-326, 330, 338, 382, 405, 447-484, 644-695, 843-879 and a plurality of said J segment oligonucleotide primers comprising sequences selected from the group consisting of 53-63, 65, 215-220, 247, 638-639, 696-697 and 699-701 to produce a plurality of rearranged DNA amplicons;
d) sequencing said plurality of rearranged DNA amplicons using high throughput single molecule sequencing to produce a plurality of sequence reads; and,
e) determining the relative quantity of and/or degree of clonality of said plurality of sequence reads in step (d) thereby determining the relative quantity of and/or degree of clonality of tumor infiltrating lymphocytes in the first sample and the second sample wherein the relative quantity number of and/or degree of clonality of tumor infiltrating lymphocytes is an assessment of prognosis of a subject receiving therapeutic treatment for a solid tumor.
2. The method of claim 1 further comprising calculating a total number of genomes in said DNA extracted from the samples in step (b) and comparing the number of rearranged DNA amplicons sequenced in step d) to the total number of calculated genomes thereby determining the relative quantity of tumor infiltrating lymphocytes.
3. The method of claim 2 wherein the total number of genomes is calculated by dividing the amount of DNA obtained in step (b) by the average weight of a single genome.
4. The method of claim 1 wherein determining the relative quantity of tumor infiltrating lymphocytes is determined by comparing the number of rearranged DNA amplicons sequenced in step d) to a level of control genes amplified in a PCR reaction using primers capable of amplifying said control genes.
5. The method of claim 4 wherein the PCR reaction is the multiplex PCR reaction of step (c).
6. The method of claim 4 wherein the PCR reaction is a separate PCR from the multiplex reaction in step (c).
7. The method of claim 1 further comprising determining a total number of unique adaptive immune receptor CDR3 bearing cells from the rearranged DNA amplicons sequenced in step (d).
8. The method of claim 7 wherein the total number of unique adaptive immune receptor CDR3 bearing cells is determined by employing the unseen species formula.
9. The method of claim 1 further comprising calculating and correcting for PCR bias based on the number of PCR cycles performed in step (c).
10. The method of claim 1 wherein the plurality of V segment oligonucleotide primers and the plurality of J segment oligonucleotide primers each contain at least one additional sequence wherein the at least one additional sequence comprises nucleotides that are not the same as or complementary to target V or J segments.
11. The method of claim 10 wherein the at least one additional sequence is one or more of a restriction enzyme recognition site, an adapter sequence for sequencing or a bar code sequence.