A method for analyzing the similarity of at least two samples using deterministic restriction enzyme site-whole genome amplification (DRS-WGA).

Abstract

This disclosure relates to a method for analyzing the similarity between at least two samples in a group of samples containing genomic DNA. [Solution] The method includes the following steps: a) Providing multiple samples containing genomic DNA. b) Performing deterministic restriction enzyme site whole-genome amplification (DRS-WGA) of the genomic DNA separately for each sample. c) Preparing a massively parallel sequencing library from each product of the DRS-WGA using a non-fragmentation sequencing adapter / WGA fusion primer PCR reaction. d) Performing low-pass whole-genome sequencing with the massively parallel sequencing library at an average coverage depth of <1×. e) Aligning the reads obtained in step d) to a reference genome for each sample. f) Extracting allele contents from multiple polymorphic loci for each sample. g) Calculating pairwise similarity scores of at least two samples as a function of the allele contents measured at the multiple loci. h) Determining the similarity of at least two samples based on the similarity scores.

Claims

1. A method for analyzing the similarity of at least two samples among multiple samples containing genomic DNA, a) A step of providing multiple samples containing genomic DNA; b) A step of performing deterministic restriction enzyme site whole genome amplification (DRS-WGA) of the genomic DNA separately for each sample; c) A step of preparing a massively parallel sequencing library from each of the DRS-WGA products using a non-fragmentation sequencing adapter / WGA fusion primer PCR reaction; d) A step of performing low-pass whole-genome sequencing with an average coverage depth of <1× using the aforementioned ultra-parallel sequencing library; e) The process of aligning the reads obtained in step d) to a reference genome for each sample; f) A process of extracting allele contents from multiple polymorphic loci for each sample; g) a step of calculating pairwise similarity scores of at least two samples as a function of the allele contents measured at the plurality of loci; h) A step of determining the similarity of at least two samples based on similarity scores. A method that includes this.

2. The method according to claim 1, wherein the low-pass whole-genome sequencing is performed with coverage <0.01×, preferably <0.05×, more preferably <0.1×, and even more preferably <0.5×.

3. The method according to claim 1 or 2, wherein the plurality of polymorphic loci include polymorphic loci with an average heterozygosity > 0.499, preferably an average heterozygosity > 0.49, more preferably an average heterozygosity > 0.4, even more preferably an average heterozygosity > 0.3, and most preferably an average heterozygosity > 0.

2.

4. The method according to any one of claims 1 to 3, wherein the plurality of polymorphic loci include >200,000 loci, preferably >300,000 loci, more preferably >500,000 loci, and even more preferably >1,000,000 loci.

5. The method according to any one of claims 1 to 4, wherein the pairwise similarity score is calculated by calculating the correlation of B allele frequencies across loci covered by at least one read of at least two samples.

6. The pairwise similarity score is calculated by determining the average match value across loci covered by at least one read from both pairs of samples, where the match value for each locus is as follows: A1) If the called alleles are the same, then 1; and B1) If the called allele is different, then 0; or A2) If the called alleles are the same, then 1; B2) If the called alleles are completely different, then 0; and C2) If there is some overlap in the called alleles, then 0.5 The method according to any one of claims 1 to 4, wherein one of the following is assigned.

7. The method according to any one of claims 1 to 6, further comprising the step of defining a group of clusters of samples that share a common characteristic selected from the group consisting of the identity of one (or more) individuals that substantially contribute to the cluster samples by DNA, or the characteristic of containing an insufficient amount of DNA, and / or the characteristic of containing highly degraded DNA or DNA of unknown origin.

8. The method according to claim 7, wherein an algorithm using the pairwise similarity scores as input assigns at least two samples to at least one cluster.

9. The method according to claim 8, wherein the algorithm is a hierarchical clustering algorithm.

10. The number of the aforementioned clusters is a) The step of selecting a number of first iterative clusters that maximize the mean silhouette score; b) A step of calculating the silhouette score of each sample belonging to the first repeating cluster for each of the first repeating clusters, wherein samples belonging to a cluster having a silhouette score lower than a fixed threshold within the range of 0.19 to 0.21 are assigned to a new cluster. The method according to claim 8, calculated by...

11. The method according to claim 10, wherein the cluster group comprises one or more identity clusters, each containing a sample with DNA derived from only one identical individual.

12. The method according to claim 11, wherein, in the presence of multiple identity clusters, the concentrations of the multiple identity clusters correspond to the number of individual DNA contributors in the multiple samples.

13. The method according to any one of claims 8 to 12, further comprising the step of defining a group of mixed identity clusters, wherein each of the mixed identity clusters includes a sample containing DNA from at least two individuals.

14. The method according to claim 13, further comprising the step of defining at least one no-call cluster, which includes a sample containing DNA of unknown origin.

15. The method according to any one of claims 8 to 14, wherein the plurality of samples include at least one reference sample, and the identity cluster group includes at least one reference cluster which includes the reference sample.

16. The method according to claim 15, wherein the at least one reference sample is a sample derived from a pregnant female parent.

17. The method according to claim 16, wherein the identity cluster group further comprises at least one kinship cluster consisting of samples derived from at least one fetus from the ongoing pregnancy of the female parent individual.

18. The method according to claim 17, wherein the related cluster is divided into a plurality of fetal clusters, each consisting of a sample containing DNA from only one identical fetus.

19. The method of claim 15, wherein the at least one reference cluster comprises samples containing DNA from only one identical individual corresponding to a victim in a forensic investigation, further comprising the step of defining at least one perpetrator cluster containing samples containing DNA from only one identical individual distinct from the victim.

20. The method according to claim 19, comprising the steps of: cluster-wise mixing DRS-WGA aliquots from multiple samples belonging to each of the at least one perpetrator clusters; preparing corresponding single-individual WGA-DNA samples for each cluster; and performing further DNA analysis on at least one of the single-individual WGA-DNA samples.

21. The method according to claim 19, comprising the steps of clusterwise integrating genetic analysis data of at least one assay type from a plurality of samples belonging to each of the at least one perpetrator clusters, and creating corresponding single-individual WGA-DNA data for each of the at least one perpetrator clusters.

22. The assay type is, a) Microsatellite analysis; b) Single nucleotide polymorphism analysis; c) Massively parallel target sequences; and d) Whole genome sequencing The method according to claim 21, selected from the group consisting of the following.

23. The method according to any one of claims 1 to 15, wherein the plurality of samples include tumor and / or normal samples.

24. The plurality of samples include at least a reference sample containing DNA derived from the female parent, and at least one other embryo sample from the plurality of samples, a) A sample containing DNA derived from an embryo from the female parent; and b) Sample containing DNA from used embryo culture medium obtained from the embryo of the female individual. The method according to claim 1 or 15, selected from the group consisting of the following.

25. The method according to claim 24, further comprising the step of performing preimplantation genetic screening of the embryo by analyzing genome-wide chromosomal abnormalities from the low-pass whole-genome sequencing data from the at least one other embryo sample, using a contamination coefficient corresponding to maternal contamination measured in the at least one other embryo sample as a function of the pairwise similarity of the at least one other embryo sample from the female parent sample.

26. The method according to claim 15, wherein the plurality of samples include at least a reference sample containing DNA derived from a female parent and at least one other sample containing DNA from a cell-free DNA sample.

27. The method according to claim 26, further comprising the step of performing non-invasive prenatal testing of the cell-free DNA sample by analyzing genome-wide chromosomal abnormalities from the low-pass whole-genome sequencing data from the at least one cell-free DNA sample using a correction factor corresponding to the fetal fraction measured in the at least one cell-free DNA sample as a function of pairwise similarity with a maternal reference sample.

28. The method according to claim 15, wherein the plurality of samples include at least a reference sample containing DNA derived from a female parent, and at least one other prenatal sample containing DNA derived from chorionic villi, amniotic fluid, or a product of conception.

29. The method according to claim 28, further comprising the step of performing prenatal testing on the prenatal sample by analyzing genome-wide chromosomal abnormalities from low-pass whole-genome sequencing data from the at least one prenatal sample using a correction factor corresponding to maternal or exogenous contamination measured in the at least one prenatal sample as a function of pairwise similarity with a maternal reference sample.

30. The method according to claim 15, particularly for cell line authentication, wherein multiple reference clusters are generated from multiple samples of DNA derived from a cell line, and the identity cluster group further comprises at least one sample derived from the cell line to be authenticated.

31. The method according to claim 15, particularly for examining an allograft, wherein the at least one reference cluster is comprised of a sample containing germline DNA derived from the transplant patient, and the identity cluster group further comprises a donor cluster comprised of a sample derived from an allogeneic donor of the transplant patient.

32. The at least one reference sample comprises the father reference sample containing only father-derived DNA, and the at least one reference cluster further comprises a father identity cluster containing the father's sample. (i) If the similarity score of the related sample to the male sample matches the related sample, then it is confirmed that he is the father, (ii) If the similarity score of a related sample to a male sample matches that of an unrelated individual, then it cannot be confirmed that the individual is the father. The method according to claim 17, particularly for non-invasive paternity testing.

33. The method according to claim 17, in particular for non-invasive assessment of molar pregnancy, wherein if the at least one sample comprises at least one circulating trophoblast cell sample, and the trophoblast cell sample similarity score to the maternal sample matches that of the unrelated sample, then a total molar pregnancy is confirmed.

34. The aforementioned at least one sample includes multiple trophoblast cell samples, (i) If the similarity score between the trophoblast cell samples exceeds the predicted 99th percentile of the predicted similarity score of the self-sample, then P1P1 homozygous paternal mole is confirmed, (ii) If the similarity scores between the trophoblast cell samples match the predicted similarity scores of the self-sample, then P1P2 heterozygous paternal mole is confirmed. The method according to claim 33.

35. The at least one sample further includes a sample from the father, and the similarity score between the trophoblast cell samples matches the predicted similarity score of the self-sample. (i) If the trophoblast cell sample similarity score for the male parent's sample matches the predicted similarity score for the self-sample, then P1P2 heterozygous paternal mole is confirmed, (ii) If the trophoblast cell sample similarity score to the male parent's sample is lower than the 1st percentile of the predicted similarity score of the self-sample, then P1P2 heterozygous paternal mole is not supported. The method according to claim 30.

36. The method according to any one of claims 1 to 6, further comprising the step of classifying a sample selected from a plurality of samples based on a predefined class using a machine learning classifier that takes the pairwise similarity scores as input.

37. The method according to claim 36, wherein the machine learning classifier is a random forest classifier.

38. A machine learning classifier, a)DLRS: derivative log ratio spread; b) R50: The percentage of WGA fragments covered by 50% of sequence reads relative to all WGA fragments covered by at least one read; c) YFRAC: fraction of reads mapped to chromosome Y; a) Aberrant: The proportion of the genome corresponding to the increase or decrease in median cytoploidy; b) Chr13: Ploidy of chromosome 13; c) Chr18: Ploidy of chromosome 18; d) Chr21: Ploidy of chromosome 21; e) RSUM: The mean absolute deviation from the nearest integer copy number level, calculated for the copy number anomalous event with the highest absolute deviation from the median of cell ploidy; f) Mix_score: RSUM z-score calculated for the copy number abnormality event with the highest absolute deviation from the median cytoploidy; and g) Deg_score: Number of small decrement events (<10Mbp, commonly seen in degraded samples) The method according to claim 36 or 37, further using as a further input at least one value measured for the low-pass whole-genome sequencing data, selected from the group including the following:

39. The method according to any one of claims 36 to 38, wherein at least one of the samples is a reference sample.

40. The method according to claim 39, wherein the at least one reference sample includes a sample derived from a pregnant female parent.

41. The method according to claim 40, wherein the plurality of samples include at least one sample classified as “related” to the mother reference, which corresponds to a sample derived from a fetus from the ongoing pregnancy of the mother individual.

42. The method according to claim 39, wherein the at least one reference sample is a sample containing DNA from the same individual corresponding to the victim in a forensic investigation, further comprising the step of clarifying at least one group of single perpetrators represented by all samples that are classified as "non-self" to the reference sample and "self" to one another, including a sample containing DNA from the same individual that is different from the victim.

43. The method according to claim 42, comprising the steps of: groupwise mixing DRS-WGA aliquots from multiple samples belonging to each of the at least one single perpetrator group; preparing a single individual WGA-DNA sample corresponding to each single perpetrator group; and performing further DNA analysis on at least one of the single individual WGA-DNA samples.

44. The method according to claim 42, comprising the step of groupwise integrating genetic analysis data of at least one assay type from multiple samples belonging to each of the at least one single perpetrator group to create the single individual WGA-DNA data corresponding to each of the at least one single perpetrator group.

45. The method according to any one of claims 36 to 39, wherein the plurality of samples include tumor and / or normal samples.

46. The plurality of samples include at least a reference sample containing DNA derived from the mother, and at least one other embryo sample from the plurality of samples is classified as "non-self" with respect to the mother reference. a) A sample containing DNA derived from the embryo of the mother individual; and b) A sample containing DNA from used embryo culture medium obtained from the embryo of the aforementioned mother. The method according to any one of claims 36 to 39, selected from the group consisting of the following.

47. The method according to claim 46, further comprising the step of performing preimplantation genetic screening of the embryo by analyzing genome-wide chromosomal abnormalities from the low-pass whole-genome sequencing data from the at least one other embryo sample, using a contamination coefficient corresponding to maternal contamination measured in the at least one other embryo sample as a function of the pairwise similarity of the at least one other embryo sample from the female parent sample.

48. The method according to claim 39, wherein a plurality of reference groups are generated from a plurality of samples of DNA derived from a cell line, the plurality of samples further comprising at least one sample derived from a cell line to be authenticated.

49. The method according to claim 39, wherein the at least one reference group comprises a sample containing germline DNA derived from a transplant patient, and the plurality of samples further comprises one donor sample corresponding to at least one sample derived from an allogeneic donor of the transplant patient.

50. The at least one reference sample further comprises a father reference sample containing only the DNA derived from the father, and the plurality of samples are (i) If a sample is classified as “self” relative to the father reference sample, then it is confirmed that the person is a father. (ii) If a person is classified as "unrelated" to the male reference sample, then it cannot be confirmed that they are the father. The method according to claim 41, further comprising a sample, particularly for non-invasive paternity testing.

51. The method according to claim 40, particularly for non-invasive assessment of molar pregnancy, wherein if the at least one sample comprises at least one circulating trophoblast cell sample, and the trophoblast cell sample is classified as “unrelated” with respect to the maternal reference, then a complete hydatidiform mole of paternal origin is confirmed.

52. The aforementioned at least one sample includes multiple trophoblast cell samples that are classified as "self" to one another, (i) If their similarity scores exceed the predicted 99th percentile of the predicted similarity score of the “self” sample, then paternal P1P1 homozygous hydatidiform mole is confirmed. (ii) If their similarity scores match the predicted similarity scores of the “self” sample, then paternal P1P2 heterozygous hydatidiform mole is confirmed. The method according to claim 51.

53. The at least one sample further comprises a sample from the father, and the father's sample is classified as "self" with respect to at least one of the plurality of trophoblast cell samples. (i) If the trophoblast cell sample similarity score for the male parent's sample matches the predicted similarity score for the "self" sample, then the paternal P1P2 heterozygous hydatidiform mole is confirmed, (ii) If the trophoblast cell sample similarity score to the male parent's sample is lower than the 1st percentile of the predicted similarity score of the "self" sample, then paternal P1P2 heterozygous hydatidiform mole is not confirmed. The method according to claim 52.

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