| 主权项 |
1. A method of determining allele profiles by identifying alleles at one or more polymorphic sites, for at least two nucleic acid samples in a plurality of nucleic acid samples, the method comprising:
(a) binning one or more of the alleles to be identified based on the alleles to be binned having the same source tag sharing number “d”, wherein said source tag sharing number “d” is determined based on a frequency of the one or more alleles to be identified, wherein if said number “d” is not an integer, then said number “d” is set to the closest positive integer that is less than “d”; provided that if “d” is less one, then “d” is set to one; further provided that if the resulting integer “d” exceeds a preset upper limit of “d”, it is replaced by said preset upper limit of “d”, wherein said preset upper limit of “d” is the lesser of: (i) a value of “d” determined by the condition that the probability of an ambiguity in the identification of said alleles occurring in “d” nucleic acid samples of the plurality of nucleic acid samples does not exceed a user-selected highest acceptable probability of an ambiguity in the identification of said alleles occurring in “d” nucleic acid samples of the plurality of nucleic acid samples; provided that the user-selected highest acceptable probability of an ambiguity is not 0 or 1 and provided that the frequency of a variant allele based on the alleles to be identified is not 0; and (ii) the maximum pool size, which is a number of nucleic acid samples that may be pooled, wherein the number is greater than one and is based on technical limitations of performing the steps of the herein method of determining allele profiles; (b) collecting aliquots from the plurality of nucleic acid samples and combining “d” number of said aliquots to form at least one nucleic acid sample pool; (c) performing a reaction in each nucleic acid sample pool to produce reaction products comprising a source tag identifying each said pool and polymorphic sites comprising said binned alleles, wherein said reaction is performed using as templates said nucleic acid samples in each said pool; (d) if “d” is less than the maximum pool size, then forming at least one pooled pool comprising at least some of the said produced reaction products from a number of pools, said number determined as the result of dividing the maximum pool size by “d”; or if “d” is equal to the maximum pool size, then proceeding to step (e) without forming pooled pools; (e) for each of the alleles to be identified, performing a second reaction in the at least one pool or pooled pool using said reaction products comprising said source tag to produce allele-specific second reaction products comprising a marker tag and a derived source tag, wherein said derived source tag is at least one of: said source tag, a copy of said source tag, or a copy of the complement of the source tag, and wherein said marker tag identifies an allele at a polymorphic site; (f) interrogating said marker tags; (g) (i) if the marker tags at a polymorphic site comprising the one or more alleles to be identified are identical, then identifying the nucleic acid samples identified by source tags or derived source tags as being homozygous for said allele of the one or more alleles to be identified; (g) (ii) if the marker tags at a polymorphic site comprising one or more alleles to be identified are different, then identifying the polymorphic site with said different marker tags; and (h) for nucleic acid samples comprising polymorphic site(s) identified in (g) (ii), repeating steps (a)-(g) as needed on said identified nucleic acid samples until all of the alleles constituting the allele profile to be determined have been determined for each of the nucleic acid samples, wherein in carrying out repetitions of steps (a)-(g) “d” is replaced by an integer “d*” that is different than “d”. |
