Analysis system for peripheral blood-based non-invasive detection of lesion immune repertoire diversity and uses of system
AssigneeGeneplus - Beijing
InventorsYuqi Wang, Yanfang Guan, Xin Yi, Tao Liu, Linjun Wu, Ling Yang
Life of this patent
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Abstract
A method for analyzing the diversity of immune repertoire of T cell receptors (TCR) or B cell receptors (BCR) of a cell-free DNA (cf-DNA) sample and of a nuclear DNA sample of peripheral blood mononuclear cell (PBMC), applicable in screening and determining the presence of lesion-infiltrating lymphocytes.
Claims18
A system for assessing immune repertoire diversity of a T cell antigen receptor (TCR) or a B cell antigen receptor (BCR) in a plasma cell-free DNA (cf-DNA) and a nuclear DNA (g-DNA) isolated from peripheral blood mononuclear cells (PBMCs), comprising the following units:
- 1) a reference sequence set construction unit that constructs a reference sequence set;
- 2) a sample preparation unit;
- 3) a library preparation and high-throughput sequencing unit;
- and
- 4) a immune repertoire bioinformatics analysis unit.
6-8. (canceled)
A primer combination for detecting TCR or BCR immune repertoire in plasma cfDNA and PMBC gDNA, wherein the sequences of the primer combination are shown in the FIG. 1 and FIG. 2.
A bioinformatics analysis unit, capable of executing the following instructions:
- 1) performing MiXCR software analysis, filtering out low-quality data, correcting PCR and sequencing errors, and identifying CDR3 sequences;
- 2) performing non-invasive lesions infiltrating lymphocytes analysis (NILILa), comprising the following contents:
- if the ranking of relative abundance of N TCRs/BCRs in plasma constitutes a collection Y(y1≤y2≤ . . . ≤yN), since a normal TCR/BCR library in a patient's plasma comes from a normal distribution population, the disease-specific TCR/BCR sub-library released from his lesion sites will cause a skewed distribution of a plasma TCR/BCR total library after entering plasma;
- supposing the probability density function of the skewed distribution is cdf:
- F (Y|θ), wherein θ is the decision parameter set of F;
- θ can be obtained by solving Equation 1 based on the principle of minimum variance, Equation 1 being described as follows:
- θ
- =
- arg
-
-
- min
- θ
-
- ∑
- i
- ∈
- Λ
-
-
- [
- g
-
- (
- y
- i
- )
- -
- g
-
- (
- F
- -
- 1
-
- (
- F
- i
- |
- θ
- )
- )
- ]
- 2
- ,
- wherein A is an index set of Y subset, represents a relative abundance of the ith TCR/BCR CDR3, g is a monotonic function that can be differentiated within the value range of Y;
- cdf is obtained by solving this equation, the expression of cdf being as follows:
- 1
- 2
- +
- 1
- 2
-
- erf
-
- {
- (
- y
- -
- μ
- )
- 2
- 2
-
- σ
- }
- ,
- a TCR/BCR frequency distribution detected in plasma can be determined according to this model probability density distribution function;
- supposing there are two thresholds ρ±, when a frequency of TCR/BCR is higher than ρ+ or lower than ρ−, the number of CDR3 is ρ±, and Equation 2 is solved, the expression of Equation 2 being as follows:
-
- ρ
- ±
- =
- F
- -
- 1
-
- (
- δ
- ±
- ∓
- ρ
- ±
- N
- |
- θ
- )
- ,
- a threshold ρ± is obtained as follows:
-
- ρ
- ±
- =
- 2
-
- σ
-
-
- erf
- -
- 1
-
- [
- ±
- (
- 1
- -
- 2
-
- ρ
- ±
- N
- )
- ]
- +
- μ
- ,
- furthermore, in order to explore more outlier TCRs/BCRs associated with lesion sites, ρ± is set to 1, a relative abundance value ρ± characterizing outlier TCRs/BCRs is calculated, and then this value can be used as a boundary of distinguishing outliers, and a frequency value corresponding to this point is called the plasma B (boundary, B) point;
- furthermore, in order to avoid an impact of a lymphocyte total library in PBMCs on results, the following chart is drawn to exclude interference from the lymphocyte total library in PBMCs:
- an abscissa is an order of a frequency of clones detected in PBMCs from high to low, and an ordinate is an order of a frequency of clones detected in plasma from low to high;
- in this chart, frequency coordinates of each clone in two samples are marked, and then two points are found:
- abscissa and ordinate values of the first point are both maximum values, and the second point has an abscissa value of 0 and an ordinate value of B value;
- these two points are connected to form a line segment which divides coordinates into two parts:
- the upper right part is a distribution area of lesions infiltrating lymphocytes, and the lower left part is a distribution area of other background clones;
- points in the upper right part are output, and are CDR3 sequences of lesions infiltrating lymphocytes.
A method for analyzing in a subject in need thereof immune repertoire diversity of TCRs or BCRs in plasma cell-free DNA (cf-DNA) and nuclear DNA (g-DNA) isolated from peripheral blood mononuclear cells (PBMCs) of said subject, the method comprising the following steps:
- 1) Constructing a reference sequence set and designing a specific amplification primer;
- 2) preparing the cf-DNA and the g-DNA from peripheral blood of the subject;
- 3) preparing and sequencing a library of PCR amplification products of the cf-DNA and the g-DNA to obtain sequence data;
- and
- 4) Analyzing bioinformatics based on the sequence data obtained in 3).
Dependencies are inferred from the claim text.