Method of Measuring Adaptive Immunity
A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.
1 . A method for determining a profile of clonotypes of recombined DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of recombined DNA sequences from said cells on a solid surface;
(c) sequencing said spatially isolated individual molecules of recombined DNA sequences to provide at least 1000 sequence reads each comprising at least 30 bp so that different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent; and
(d) determining levels of different clonotypes from said sample to generate said profile of clonotypes of recombined DNA sequences.
2 . A method for determining a profile of clonotypes of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) spatially isolating individual molecules of genomic DNA from said cells on a solid surface;
(c) sequencing said spatially isolated individual molecules of genomic DNA to provide at least 1000 sequence reads each comprising at least 30 bp so that different clonotypes of said sample are determined from such sequence reads with a confidence of at least 99.9 percent; and
(d) determining levels of different clonotypes from said sample to generate said profile of clonotypes of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences.
3 . The method of any one of claims 1 - 2 , wherein the individual molecules comprise a repertoire of T-cell receptor genes and/or B-cell receptor genes.
4 . The method of any one of claims 1 - 3 , wherein the solid surface is provided by a glass slide or beads.
5 . The method of claim 1 , wherein the molecules of recombined DNA sequences are amplified.
6 . The method of claim 2 , wherein the molecules of genomic DNA are amplified.
7 . The method of any one of claims 1 to 2 , wherein said sequencing comprises sequencing by synthesis using reversibly terminated labeled nucleotides.
8 . The method of any one of claims 1 to 2 , wherein said step of sequencing includes providing a number of said sequence reads which depends on base quality scores of said sequence reads.
9 . The method of any one of claims 1 to 2 , wherein said step of sequencing includes providing at least 10,000 said sequence reads each comprising at least 30 bp.
10 . A method for determining a diversity of clones of recombined DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) hybridizing individual molecules of recombined DNA sequences from said cells to spatially isolated regions of a solid surface;
(c) sequencing said individual molecules of recombined DNA sequences to provide at least 1000 sequence reads, wherein each sequence read comprises at least 30 base pairs, such that each sequence comprising a unique clone is identified; and
(d) determining a frequency of each unique clone from said sample to generate said diversity of clones of recombined DNA sequences.
11 . A method for determining a diversity of clones of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences in T-cells and/or B-cells comprising:
(a) obtaining a sample from a subject comprising T-cells and/or B-cells;
(b) hybridizing individual molecules of recombined DNA sequences from said cells to spatially isolated regions of a solid surface;
(c) sequencing said individual molecules of recombined DNA sequences to provide at least 1000 sequence reads, wherein each sequence read comprises at least 30 base pairs, such that each sequence comprising a unique clone is identified; and
(d) determining a frequency of each unique clone from said sample to generate said diversity of clones of recombined T-cell receptor gene and/or immunoglobulin gene DNA sequences.
12 . The method of claim 10 or 11 , wherein the individual molecules comprise a repertoire of T-cell receptor genes and/or B-cell receptor genes.
13 . The method of claim 10 or 11 , wherein said solid surface is provided by a glass slide or beads.
14 . The method of claim 10 , wherein said molecules of recombined DNA sequences are amplified.
15 . The method of claim 11 , wherein the molecules of recombined DNA sequences comprise genomic DNA and are amplified.
16 . The method of claim 10 or 11 , wherein said sequencing comprises sequencing by synthesis using reversibly terminated labeled nucleotides.
17 . The method of claim 10 or 11 , wherein said sequencing comprises providing a number of said sequence reads which depends on the quality of said sequence reads.
18 . The method of claim 10 or 11 , wherein said sequencing comprises providing at least 10,000 said sequence reads each comprising at least 30 base pairs.