WO1998058529A2

WO1998058529A2 - Genetic compositions and methods

Info

Publication number: WO1998058529A2
Application number: PCT/US1998/012930
Authority: WO
Inventors: Robert J. Lipshutz; Mark Chee; Jian-Bing Fan; Anthony Berno
Original assignee: Affymetrix, Inc.
Priority date: 1997-06-24
Filing date: 1998-06-22
Publication date: 1998-12-30

Description

GENETIC COMPOSITIONS AND METHODS

BACKGROUND OF THE INVENTION The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution generating variant forms of progenitor sequences (Gusella, Ann. Jev. Biochem. 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.

Several different types of polymorphism have been reported. A restriction fragment length polymorphism (RFLP) means a variation in DNA sequence that alters the length of a restriction fragment as described in Botstein et al . , Am. J. Hum. Genet. 32, 314-331 (1980) . The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment . RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; W090/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait. Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra- nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis (US 5,075,217; Armour et al . , FEBS Lett . 307, 113-115 (1992); Horn et al . , WO 91/14003; Jeffreys, EP 370,719), and in a large number of genetic mapping studies.

Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms occur in protein- coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include β-globin (sickle cell anemia) and CFTR (cystic fibrosis) . Other single nucleotide polymorphisms occur in noncoding regions . Some of these polymorphisms may also result in defective protein expression

(e.g., as a result of defective splicing) . Other single nucleotide polymorphisms have no phenotypic effects.

Single nucleotide polymorphisms can be used in the same manner as RFLPs, and VNTRs but offer several advantages. Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms. Also, the different forms of characterized single nucleotide polymorphisms are often easier to distinguish that other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers) .

Despite the increased amount of nucleotide sequence data being generated in recent years, only a minute proportion of the total repository of polymorphisms in humans and other organisms has so far been identified. The paucity of polymorphisms hitherto identified is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymorphisms might be to sequence the same stretch of oligonucleotides in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.

SUMMARY OF THE CLAIMED INVENTION The invention provides nucleic acid segments of between 10 and 100 bases from a fragment shown in Table 1, column 1 including a polymorphic site. Complements of these segments are also included. The segments can be DNA or RNA, and can be double- or single-stranded. Some segments are 10- 20 or 10-50 bases long. Preferred segments include a diallelic polymorphic site. The base occupying the polymorphic site in the segments can be the reference (Table 1, column 3) or an alternative base (Table 1, column 5) . The invention further provides allele-specific oligonucleotides that hybridizes to a segment of a fragment shown in Table 1, column 8 or its complement. These oligonucleotides can be probes or primers. Also provided are isolated nucleic acids comprising a sequence of Table 1, column 8, or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in Table 1, column 3.

The invention further provides a method of analyzing a nucleic acid from an individual. The method determines which base is present at any one of the polymorphic sites shown in Table 1. Optionally, a set of bases occupying a set of the polymorphic sites shown in Table 1 is determined. This type of analysis can be performed on a plurality of individuals who are tested for the presence of a disease phenotype . The presence or absence of disease phenotype can then be correlated with a base or set of bases present at the polymorphic sites in the individuals tested.

The invention further provides computer-readable storage medium for storing data for access by an application program being executed on a data processing system. Such a medium comprises a data structure stored in the computer- readable storage medium, the data structure including information resident in a database used by the application program. The data structure includes a plurality of records, each record of the plurality comprising information identifying a polymorphisms shown in Table 1.

The invention further provides a signal carrying data for access by an application program being executed on a data processing system. A data structure is encoded in the signal. The data structure includes information resident in a database used by the application program. Such information includes a plurality of records, each record of the plurality comprising information identifying a polymorphism shown in Table 1.

BRIEF DESCRIPTION OF THE FIGURES Figs. 1A and IB depict computer systems suitable for storing and transmitting information relating to the polymorphisms of the invention.

DEFINITIONS An oligonucleotide can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred oligonucleotides of the invention include segments of DNA, or their complements including any one of the polymorphic sites shown in Table 1. The segments are usually between 5 and 100 bases, and often between 5-10, 5-20, 10-20, 10-50, 15-50, 15-100, 20-50 or 20-100 bases. The polymorphic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in Table 1. Hybridization probes are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al . , Science 254, 1497-1500 (1991).

The term primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes . The term primer pair means a set of primers including a 5 ' upstream primer that hybridizes with the 5 ' end of the DNA sequence to be amplified and a 3 ' , downstream primer that hybridizes with the complement of the 3¹ end of the sequence to be amplified.

Linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers.

Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphic locus may be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified allelic form is arbitrarily designated as a the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms.

A single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations) .

A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine . A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.

Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridizations.

An isolated nucleic acid means an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition) . Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) .

Linkage disequilibrium or allelic association means the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles a and b, which occur equally frequently, and linked locus Y has alleles c and d, which occur equally frequently, one would expect the combination ac to occur with a frequency of 0.25. If ac occurs more frequently, then alleles a and c are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles.

A marker in linkage disequilibrium can be particularly useful in detecting susceptibility to disease (or other phenotype) notwithstanding that the marker does not cause the disease. For example, a marker (X) that is not itself a causative element of a disease, but which is in linkage disequilibrium with a gene (including regulatory sequences) (Y) that is a causative element of a phenotype, can be used detected to indicate susceptibility to the disease in circumstances in which the gene Y may not have been identified or may not be readily detectable.

The present invention includes the use of any of the polymorphic forms shown in Table 1 as a means to determine susceptibility to a phenotype resulting from an allele or marker in linkage disequilibrium with such polymorphic forms.

DESCRIPTION

I . Novel Polymorphisms of the Invention

The novel polymorphisms of the invention are listed in Table 1. The first column of the Table lists the names assigned to the fragments in which the polymorphisms occur. The fragments are all human genomic fragments. SGC, TIGR and WI respectively stand for Stanford Genome Center, The Institute for Genome Research and the Whitehead Institute. The sequence of one allelic form of each of the fragments (arbitrarily referred to as the prototypical or reference form) has been previously published. These sequences are listed at http://www-genome.wi.mit.edu/ (all STS ' s (sequence tag sites)); http://shgc.stanford.edu (Stanford STS's); and http://ww.tigr.org/ (TIGR STS's). The Web sites also list primers for amplification of the fragments, and the genomic location of fragments. Some fragments are expressed sequence tags, and some are random genomic fragments. All information in the websites concerning the fragments listed in Table 1 is incorporated by reference in its entirety for all purposes. The second column lists the position in the fragment in which a polymorphic site has been found. Positions are numbered consecutively with the first base of the fragment sequence as listed in one of the above databases being assigned the number one. The third column lists the base occupying the polymorphic site in the sequence in the data base. This base is arbitrarily designated the reference or prototypical form but is not necessarily the most frequently occurring form. The fifth column in the table lists the alternative base(s) at the polymorphic site. The eighth column of the Table lists about 15 bases of sequence on either side of the polymorphic site in each fragment. The indicated sequences can be either DNA or RNA. In the latter, the T's shown in the Table are replaced by U's. The base occupying the polymorphic site is indicated in EUPAC-IUB ambiguity code. The fourth and sixth columns of the table show the frequency with which reference and alternative alleles occur at a polymorphic site. The seventh column in the table indicates the population frequency of heterozygotes of the polymorphic site. Also provided is a nucleic acid encoding hepatic lipase containing a polymorphism. The sequence is CTTCGAGAGAGATTGMACAGATTCCTGGAAG . Table 1

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-16260 59 G 0.79 T 0.21 0.34 GATTCAAGAAGAAAAKACCCAGAGTTTCACA

WI- 16260 86 G 0.79 A 0.21 0.34 CACAATATAGGTAGCRATAACCAGGTCTCAC

WI-16303 65 A 0.93 G 0.07 0.13 GGTCACTGCAGCCCCRTCTGTATTAGGGAGC

WI-16398 90 T 0.36 C 0.64 0.46 TCCATGATATTTTCAYAGCAACTAGTATATA

WI- 16403 69 T 0.71 C 0.29 0.41 TCAATTTTTAACACTYCTTTTTATATAGGGA

WI-16406 24 C 0.86 T 0.14 0.24 GCTACAGAAAGAAGGYGGTTTTATTTTCTTT

WI-16543 67 G 0.50 T 0.50 0.50 ACATTTGGGTTTTGGKAAGTCCCCTGTAATG

WI- 16632 71 A 0.44 G 0.56 0.49 CTACTTTGGAGCCCTRAGGAGTTTTTAGAGA

WI-16644 42 G 0.25 A 0.75 0.38 GCTCATTTTGATTACRGGTATACATGAAGTA

WI-16739 57 G 0.44 A 0.56 0.49 TTTGCCATCACAAGCRTTATAGGGAATAATG

WI-16782 96 C 0.69 T 0.31 0.43 GTCTCACTGTAAGGAYGATGGAGGAACAGAA

WI-16783 64 A 0.75 G 0.25 0.38 TGTCTTTTACCTGAGRCTAATAAGGATTGAA

WI-16816 124 A 0.75 G 0.25 0.38 CCATTGTTGGGGTTARACTGTCCTGAACAAA

WI-16824 47 T 0.25 C 0.75 0.38 TGGTGCTGCAGCTGTYGTTCTTATGAAGAAG

WI- 16824 83 G 0.75 A 0.25 0.38 AGCTGATAAACGTGGRCTTACACCTTTAGCA

WI-16857 47 G 0.13 A 0.88 0.22 GCAGCTAATGGCAATRCTAGTGGTCTTCCCA

WI- 16879 79 C 0.88 T 0.13 0.22 AGGCCATATTTCCCAYATAGGACTCTAGTTC

Fragment Position "Ref "Freq "Alt "Freq "H" "Sequence Tag" Allele" (P)" Allele" ( r

WI-16882 99 A 0.56 G 0.44 0.49 TGCCACGTCTCTGACRGCGATTTACCTGACA

WI-16888 70 G 0.38 A 0.63 0.47 ACTTTGGGCAGGTTCRTTAAATTTGGTCAAT

WI-16905 75 C 0.88 T 0.13 0.22 GGCCTGTGTTGTTCAYCCCACTGCCTAGAAG

WI-16910 74 G 0.75 A 0.25 0.38 AAGATGGCGCTAGAARGTATCTGTTATAGAA

WI-16918 93 C 0.44 T 0.56 0.49 CATTAACACCAGCACYGATGCCACTTCTGTA

WI- 16947 58 C 0.31 G 0.69 0.43 GAAATAGGCCTGGAGSACAGGATTTGGCTGA

WI-16947 127 A 0.38 C 0.63 0.47 AAAGCAGACCTGGGGMCCACGGGCAATCACA

WI-16966 43 T 0.88 C 0.13 0.22 CATAACAACCTAATAYCTTAACTTGGTCCAA

WI-16992 46 G 0.38 A 0.63 0.47 CAGAAGTACACTGTCRCCCTCATCTGAGATG

WI- 16992 60 T 0.44 G 0.56 0.49 CGCCCTCATCTGAGAKGTGTAGGACTGTAAG

WI-16995 55 T 0.25 C 0.75 0.38 GAGGTAAATAGTATTYACGGCTGGAAATCAA

WI-17010 23 T 0.81 C 0.19 0.30 ACAGGAAAAGCCATGYATGACATTCAAAACA

WI-17021 62 T 0.88 A 0.13 0.22 AGCCTATAACTACTCWGCAGCTGCCACTAAC

WI-17040 94 T 0.44 C 0.56 0.49 ATCATCTCAAGCCAGYCATCACTGAATAAGC

WI- 17044 47 G 0.69 T 0.31 0.43 GGATTAACGTATAGGKTCTTAAACAAGGGGA

WI-17065 90 T 0.31 C 0.69 0.43 GAAAAGCATAAACTTYAGGATTTCATTGTCT

WI-17066 32 A 0.38 C 0.63 0.47 CCAACATCACTGTTTMATTCCAGAACATTTT

WI-17074 86 T 0.94 G 0.06 0.12 CTCCTACACAGGCCTKCTACATAGGAGTATA

WI-17104 108 T 0.88 C 0.13 0.22 GGTTTCCAGACGGCTYTCTCTTTGTTAAGAA

Fragment Position "Ref "Freq "Alt "Freq "-ET "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-17108 74 C 0.81 T 0.19 0.30 TCTCAAAGTAAACACYGGGAGCATATGATAA

WI-17114 37 T 0.44 C 0.56 0.49 CAAGGACTTTGTTTTYGTCTCTTCACTCTGC

WI-17136 33 C 0.94 G 0.06 0.12 ATGTCCCTAAAATGTSATTCAACATATATGC

WI-17149 48 C 0.44 G 0.56 0.49 TTGAAGGAGGAACATSTCATGCACGTGCGTG

WI-17149 79 T 0.31 C 0.69 0.43 GAAACCCAATTGTCAYGTGTATGAACTACAA

WI-17150 76 T 0.38 G 0.63 0.47 GATAGTCTTCCTCTTKCATATCTTCCAGGAT

WI-17156 54 G 0.81 C 0.19 0.30 TTAGATATCTCCCATSTTCCACAGAATCAAA

WI-17163 43 A 0.75 G 0.25 0.38 AATAACAATAACGTTRAAGGCAAAAGCAAGA

WI-17177 23 A 0.94 G 0.06 0.12 CATATCCAACCAACCRTCCATCCCCACCTGT

WI-17178 127 T 0.88 C 0.13 0.22 TCCCTCATGAGGAGCYAGAAGCAGTTGAAAA

WI-17180 47 T 0.75 C 0.25 0.38 AGAGAATCCTGCACTYCCCAAGTCTCGTCGC

WI-17180 81 C 0.94 G 0.06 0.12 GGCTTCAACAATTACSAACATCTTGCCCATT

WI-17197 67 G 0.56 A 0.44 0.49 AGTAGCTGGGGCTACRGGTATGCACCACCTC

WI-17198 38 A 0.75 C 0.25 0.38 CCTTGTCCCTAGTTTMTAATTTCTCAGTGGA

WI-17347 50 A 0.25 G 0.75 0.38 AGAACTTCTCAGCCTRGTAGCACAAGTGGAT

WI-17387 55 C 0.81 G 0.19 0.30 CAGATTGAAGAAAAASAATATTAGTAGTTAC

WI- 17470 83 A 0.69 G 0.31 0.43 CGTCCCGCCAGCCCTRTCGGCCTCGTCACTG

WI-17519 55 T 0.38 C 0.63 0.47 TAGCTAATGAATGCAYAGAGTATTGCCTGCA

WI-17581 86 T 0.13 C 0.88 0.22 CCAGTTATTTGATAAYGATAGAACCCAACTA

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-17581 99 C 0.69 T 0.31 0.43 AATGATAGAACCCAAYTAGGCGCAATTTACA

WI-17596 86 A 0.63 G 0.38 0.47 TGTGTAAACACTCCCRATATTGTCGATTTCT

WI-17623 46 T 0.94 C 0.06 0.12 AATGGTGGGCACATTYGCATGTGCTTACTGG

WI-17675 103 T 0.44 C 0.56 0.49 TTTGGATGGTGACTTYCCTGGGTGGTTCCCC

WI-17687 107 C 0.81 G 0.19 0.30 AAAAAGGTTGGGGAASTGCTGGTCGGTACAA

WI-17690 63 G 0.69 A 0.31 0.43 TTTCTAGCTGTGTTTRATTTGGCTTCCCTAT

WI-17690 79 A 0.63 G 0.38 0.47 ATTTGGCTTCCCTATRGATTCAGGACCCATA

WI-17724 50 T 0.81 C 0.19 0.30 TGGGCCCTCCCTGTCYGGACACTGCCAACCC

WI-17730 39 A 0.44 C 0.56 0.49 AAGTGAAGTGCTATTMGTTACATCATACCAA rv>

WI-17730 68 T 0.94 C 0.06 0.12 AAGTGTACATACTGTYCACATGATTTATGGC

WI-17800 29 C 0.88 G 0.13 0.22 CAAGAGAAACTCACTSAAGACTGGGATTAAT

WI-17835 30 G 0.38 A 0.63 0.47 TATTGTGCTTTCTTGRGCCTGTTTCCTATAC

WI-17857 34 T 0.44 G 0.56 0.49 CTGGGATGACTTTCCKATTCTACATCAAGTA

WI-17860 121 T 0.81 A 0.19 0.30 CCAGCAAAGCAAATAWCCGACTGACTGCTCC

WI-17866 43 A 0.63 T 0.38 0.47 CTTCTCAAAATTGTTWTTTGTGTGATTAGTG

WI-17892 76 T 0.88 C 0.13 0.22 GTTTGAGATCACATAYCTGTCTCACTAGTCT

WI-17904 50 A 0.31 G 0.69 0.43 CAATAAAATGAACACRTACGGGAATTACTAT

WI-17982 98 C 0.25 T 0.75 0.38 ATAACTCCTAAAAGCYGGAAGGAGTTATTAT

WI- 17993 118 A 0.94 C 0.06 0.12 CTGTCCCTGTAATGTMCTGCTGAGAGTCCAC

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-17996 84 A 0.13 G 0.88 0.22 AGGCGAAGGGAACAGRGCTGCCCATGTGCCT

WI-18012 22 T 0.44 A 0.56 0.49 TGGGTCAGCTCCTTCWTAATGGCCTGAAGGT

WI-18012 46 T 0.38 C 0.63 0.47 TGAAGGTCATCTCCTYTCAACTTTCCAGACT

WI-18012 112 C 0.50 T 0.50 0.50 CCACTTTTGCCCCTTYGTGAAGTGTTTCCTG

WI-18012 113 G 0.56 A 0.44 0.49 CACTTTTGCCCCTTCRTGAAGTGTTTCCTGA

WI-18012 117 A 0.31 G 0.69 0.43 TTTGCCCCTTCGTGARGTGTTTCCTGATACA

WI-18041 24 A 0.75 C 0.25 0.38 AAAAGGTGCTCTTCCMGTTTCTAACTCCCTG

WI-18052 50 T 0.31 C 0.69 0.43 TTTCATGTACGAATCYTGGTTACACATCTTA

WI-18052 67 A 0.31 G 0.69 0.43 GGTTACACATCTTAGRACAGCAGAGCTGCCT

WI-18054 46 G 0.25 A 0.75 0.38 AGTGGGGGAGTAAAARTGGAAGCAGGGTGAC

WI- 18064 54 G 0.81 A 0.19 0.30 AAGCTGTATTTCAGARGAATGTCACAATCAT

WI-18068 89 G 0.94 C 0.06 0.12 ATAAAAGTAAGACCASATAAAAATACCTATG

WI-18070 28 A 0.88 C 0.13 0.22 ACTCAGAGTGTGTATMATATTAACACATGAA

WI-18080 41 T 0.19 C 0.81 0.30 TCAAACTAGTCTCTCYTTGTAATTAAAATCT

WI-18080 65 G 0.38 A 0.63 0.47 AAAATCTACTATGCCRTGTTTGACTTTTATC

WI- 18086 63 G 0.06 A 0.94 0.12 AGAAAGCATACTTCTRTGGCTTTGTTACACG

WI-18115 70 C 0.88 T 0.13 0.22 CTTTGGTATTCCCTTYCTTTGGTATGAAAGA

WI-18115 71 C 0.88 T 0.13 0.22 TTTGGTATTCCCTTCYTTTGGTATGAAAGAC

WI-18136 78 A 0.94 G 0.06 0.12 CTTTAGGTAATTTGCRTAAGAACAATAAAAG

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-18169 115 A 0.44 G 0.56 0.49 TCTTTCCGGAAGCTCRTGGAGCACAAGCAGA

WI-18181 100 A 0.63 C 0.38 0.47 CACTCCCTTCAGATCMCAAAAGCTTAACAAA

WI-18190 62 G 0.75 A 0.25 0.38 GAAGCTAATCATGGARGCAAGCTCCCTGGAG

WI-18215 78 G 0.75 A 0.25 0.38 AGAGTTCCTGCCCTCRGTGTGCGGGGGGAGA

WI-18232 60 T 0.75 A 0.25 0.38 TGTGATACACTTAAGWGAACCCCTGAAAACC

WI-18242 30 G 0.88 A 0.13 0.22 TAATCGTAACATACTRGAAAGCTGTTACAGT

WI-18266 97 C 0.38 T 0.63 0.47 TGGACTATCTTCAAAYTGCACAAATGATGCA

WI-18266 124 T 0.13 C 0.88 0.22 TGCATGAATCCACATYTGAGACCCGCAACTC

WI-18312 73 A 0.75 G 0.25 0.38 ATTGTTATTTCAAATRTATCTTCTGCTCCCT

WI-18327 104 G 0.44 A 0.56 0.49 TTCGTTAGGCTAGTTRGCTGAGCCATTGTAT

WI-18330 49 G 0.63 A 0.38 0.47 AAATCAGGGATAAGARCTGAGGAACAAGAGG

WI-18357 89 C 0.63 G 0.38 0.47 AGCCCTTAGCATCAASTCATCTTCAGTCTTT

WI-18369 58 G 0.88 A 0.13 0.22 ATCTGTCACACAATCRAAATGGATAAGGCCT

WI-18387 57 A 0.63 G 0.38 0.47 TTGGTGACCCCATACRTTTGTGGTCACATGC

WI-18420 38 C 0.19 T 0.81 0.30 GGAAAATGGGAAGAAYAGAGTGAAATTAAAG

WI-18420 108 T 0.38 C 0.63 0.47 TCAAAAAAAATCAAAYGCTTATAGCAATGCT

WI-18425 81 A 0.13 C 0.88 0.22 TCCTAGACAGATTCAMTGCACACAACAACAG

WI-18449 129 C 0.38 T 0.63 0.47 CTCTAAGTGGGACTAYTCTGGATACAGTCAG

WI-18457 120 T 0.94 C 0.06 0.12 ACATTGGGGCCACAGYAAATAGGCTAAAAGG

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

WI-18462 39 A 0.56 G 0.44 0.49 CAATGGCAGAGGTGARTAGAAACCATCTCAA WI- 18476 60 C 0.56 T 0.44 0.49 GGTGGGGGTGCGAGGYGGTCACTCCCATCGT WI-18491 109 G 0.69 A 0.31 0.43 AAATCCCAGAATGACRGGATTACAAGAAAAT WI-18517 87 C 0.81 T 0.19 0.30 GAATCAGCAGCCTGAYTGTTGCACTTGTCCA WI-18533 59 T 0.44 G 0.56 0.49 TCCCCGAGATTTTCTKCTTTATTTTATATTT WI- 18533 91 T 0.56 C 0.44 0.49 CATTTTTCATCCTAAYTTACTGAAGCCATTT WI-18612 37 A 0.56 G 0.44 0.49 CAAGTTTGGAAATGCRTATTTGCAAGCAGCA WI-18640 121 T 0.44 C 0.56 0.49 TGGGGGGGTGCAGAGYGTGTCCTCTTCAGTG WI- 18668 76 C 0.19 T 0.81 0.30 AAAACTAGGCAAAAAYAGCAAAAAGTGCAGT

TIGR-A003M18 29 A 0.75 G 0.25 0.38 AGATGAGGTTTTCCTRTGTTGGCCAGGATGG

TIGR-A003P30 117 C 0.94 G 0.06 0.12 TTTAAAGCAGTGTCASACTGGCTGCCTGAAG

TIGR-A004S34 156 C 0.25 T 0.75 0.38 CCTCATTCCTATAAAYCTTTAACAAAAACAG

TIGR-A004T44 69 G 0.81 A 0.19 0.30 AACCAAAATGATTGARTATGATAAAGAATTT

TIGR-A004T44 97 A 0.75 C 0.25 0.38 TTTTGCATGGCGATTMAAATAGAAAACCTAT

WI-18673 29 A 0.00 G 1.00 0.00 GTTTTAATTGCAAACRACTTAATTTACAGCA

WI-18680 75 T 0.50 C 0.50 0.50 CTCTAGCATCTGGAAYGCTCCGTTGTATATT

WI-18694 41 A 0.56 T 0.44 0.49 AGCCAGCTCTGACTTWCTCTCTGTTTCTGTC

WI-18704 99 A 0.56 C 0.44 0.49 TTCTCCGAGGGGTACMCCAGCAGGGCCTTCA

TIGR-A004V08 60 T 0.88 C 0.13 0.22 ACAGGCATTCTCTTAYGCCTTTTGTGGGAAG

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

TIGR-A004V26 125 A 0.94 G 0.06 0.12 ATTATCTTCACATGARAAGGTTTCAGTTTAT

TIGR-A004V28 29 A 0.38 G 0.63 0.47 TGTGGGTGCGATCTCRGCTCACTGCAACCTC

TIGR-A004X20 25 T 0.31 C 0.69 0.43 TTCTCTTCTGTAGGAYGTCTCCATGTTACAG

TIGR-A004X30 26 T 0.44 C 0.56 0.49 TAGAGTAGAACCCACYACTCTAGTAATACTT

TIGR-A004Z04 102 T 0.50 G 0.50 0.50 TGGGTATGCAAAACTKTTGCTTTCATGAAAT

TIGR-A004Z19 85 C 0.88 T 0.13 0.22 CATTTTTTTCTTTTTYTCTTCCCGATGACCA

TIGR-A004Z42 89 C 0.88 T 0.13 0.22 GGGAGGTAGGAGACTYGGACCGGCAGCCCTG

TIGR-A005D17 79 G 0.63 C 0.38 0.47 GGGAAACCCAGCAAGSCTGTCTAGATTCTTC

TIGR-A005D17 81 T 0.56 C 0.44 0.49 GAAACCCAGCAAGGCYGTCTAGATTCTTCTT

TIGR-A005D44 97 G 0.69 T 0.31 0.43 TAAAACTGTTACACTKTTTTGTTGGCTTTAA

SGC30018 77 C 0.69 T 0.31 0.43 GCACATACTTCAGGCYTGCGGCACCACCCCA SGC30036 42 T 0.75 C 0.25 0.38 TAGACAGAGGCATTAYTTTTGAAGATCTTTT SGC30050 103 A 0.31 G 0.69 0.43 CCAGAAAGCTTTACCRTCTGTCAGTTAAGCT SGC30055 32 A 0.56 G 0.44 0.49 ATCTTCAGGATAGGTRATAACAGTGTGAAGG SGC30072 28 C 0.50 T 0.50 0.50 CTTTATTTTTGGACAYGTAGCATGTTTTAAC SGC30076 97 C 0.75 T 0.25 0.38 GGTCACTTTGGGGCCYGGCGTGGGCAGAGCC SGC30117 96 A 0.50 G 0.50 0.50 GCAGTCACAATGTACRAAAATGTGACAAGAT SGC30122 74 A 0.25 G 0.75 0.38 GCAGAACTTAAACACRGAGCATTTATTGTTA SGC30126 61 T 0.94 C 0.06 0.12 AGTGAATTCAACAGTYAATGCACATGCATAC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag" Allele" (P)" Allele" (Q)"

SGC30150 101 C 0.94 T 0.06 0.12 ATCATTCTCTCTTCTYTTCACATGGTGTACT

SGC30160 57 A 0.88 G 0.13 0.22 CACAGCCCTGCCCCCRTCTTGAGATTCAGAA

SGC30207 50 A 0.81 G 0.19 0.30 TCTACATTCTGAATARAGTACATAATGGGAT

TIGR-A005E31 27 G 0.63 A 0.38 0.47 TATAACCAGGCCTCTRCTCACAGCTGTACTG

TIGR-A005E39 182 G 0.50 C 0.50 0.50 GGATGTCTTCTATTGSGGATGTCTTCTATTT

TIGR-A005E42 42 A 0.44 G 0.56 0.49 CTGCACCTTACAGAGRCTCAATTTCCCCTGA

TIGR-A005E46 76 A 0.88 G 0.13 0.22 GACTCGGTGCTTTACRTACATTACCTCACAG

SGC30222 128 A 0.75 T 0.25 0.38 CCCACCATACTGGTTWTTCCGGTACTGTTTT

SGC30272 71 T 0.69 C 0.31 0.43 GTTTCTACCCCCAATYCATTACAGTCAAATT

SGC30306 33 G 0.31 A 0.69 0.43 ATTTAATAATTTATCRCATTACAGTAGCATC

SGC30349 71 A 0.56 G 0.44 0.49 ATCCTTATCTGCACARCCATTGAAGAAAAAA

SGC30371 37 T 0.94 C 0.06 0.12 GGCAAATATGCTCTAYAAAAGAATGATCAAT

SGC30374 82 T 0.81 C 0.19 0.30 TGTCTGGCATTCTTTYGTGGGGCTGTTTTTC

SGC30386 35 C 0.69 A 0.31 0.43 GGCAACTATGTGCAGMAACAATCTGATGGGC

SGC30386 46 T 0.38 C 0.63 0.47 GCAGCAACAATCTGAYGGGCAGTCCAAACTT

SGC30404 101 A 0.75 C 0.25 0.38 AAGCTCCAGAGGCAGMGCTTACAGGAGGGGA

SGC30417 22 A 0.13 C 0.88 0.22 GCTATGTTTCCCAGGMTGGTCTTGAGCTCCT

SGC30417 61 A 0.31 C 0.69 0.43 ACAATCCTCCTTCCTMAGCCTCCTAAAGTGC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC30417 69 T 0.31 C 0.69 0.43 CCTTCCTAAGCCTCCYAAAGTGCCAGGATTA

SGC30422 24 G 0.44 A 0.56 0.49 CACTTCTGGAGGCTGRGAAGTCTAAGATTGA

SGC30515 56 A 0.56 G 0.44 0.49 CAGTACAAAGTCTGTRATCCAGGAAGTGACC

SGC30535 23 T 0.94 A 0.06 0.12 TCCACCTGACCTGCAWCAACAGCCCAGTTAT

SGC30540 52 6A 0.56 G 0.44 0.49 TGTTAAAAACAACACRTCTGTCACTTGCAGA

SGC30587 74 T 0.94 G 0.06 0.12 AATAGTCTGGCCATTKGACTAACCAGTTCTA

SGC30593 72 G 0.31 A 0.69 0.43 AGATGTGAGAGACGCRTCTCTGTACAGGAGC

SGC30598 70 T 0.81 C 0.19 0.30 GATTTTCTCAGGCCTYTTTTGGATACCTTTA

SGC30610 99 A 0.50 T 0.50 0.50 CATTTCAGTCCAAGAWAACCTTCCTCAAATT

SGC30612 39 A 0.88 G 0.13 0.22 AAGTTTGGGTTTCTTRCTGAAATTTCCATGA

SGC30622 32 T 0.63 C 0.38 0.47 CTGTTACGTCTTTCCYATTATATTTATCTTG

SGC30669 39 A 0.94 G 0.06 0.12 CACAGAGACTGTCTCRGAGACGGGCACAGAA

SGC30678 30 G 0.44 C 0.56 0.49 AATTCCCTTGGTGGGSGGGGGGGGGTGAGAT

SGC30689 58 G 0.81 A 0.19 0.30 TCATCAGAACCCCACRGTACTTGGAGTACCT

SGC30719 53 G 0.69 A 0.31 0.43 AGCATCATTGTCACTRGCTAACTCCTCAAAT

SGC30720 85 T 0.63 C 0.38 0.47 CCATCTACAAAAGATYTCTCATTGAGGCCTC

SGC30754 66 T 0.81 C 0.19 0.30 CATGTTTCTGTTTAAYTCTCTTATGTGTTAT

SGC30775 58 A 0.94 G 0.06 0.12 ATTCCAGCAGGTGCCRTTATTTTCACTTGGT

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag" Allele" (P)" Allele"

SGC30813 103 C 0.75 T 0.25 0.38 GCCAGCTTACAGGCTYACAGAAGAATGAGAC

SGC30827 121 G 0.94 C 0.06 0.12 CTACATAGGGATAAASAGCTCAGTATCTGGA

SGC30890 87 C 0.63 T 0.38 0.47 GTTGTCCAGCCAACAYGGAGGTGATTTTGGT

SGC30895 72 T 0.31 C 0.69 0.43 AATTTGTGTCGATGCYCTGTGTCCTCCGTCC

SGC30914 75 T 0.88 C 0.13 0.22 CTATAAGTGCATTTTYATAATGGGGATTTTC

SGC30914 95 T 0.56 G 0.44 0.49 TGGGGATTTTCTGCTKAACTGCCCACTGATT

SGC30938 80 A 0.38 G 0.63 0.47 ATGCAGGAGGGTGGCRAGAGGGGCCGAGATT

SGC30940 103 C 0.94 T 0.06 0.12 AGCTGGCTTTGTAGTYGTTCAGGCCCATTGA

SGC30955 69 A 0.81 G 0.19 0.30 TACTCAAGTGTGAATRGATTTTATTAGTTGT

SGC30985 75 A 0.75 G 0.25 0.38 AGGACTCTGCATTGTRATTAAGTTTATTAAT

SGC31224 47 A 0.88 G 0.13 0.22 AGCATGGCTAAAACGRTAAAGATGGGAATCA

SGC31233 85 A 0.63 G 0.38 0.47 AACTTATAACCTCACRCGCTTGTTTCACAAA

SGC31250 79 T 0.75 G 0.25 0.38 TAAGGCCTAAGGAATKAGGGGCAGGGGGCGA

SGC31279 42 G 0.56 A 0.44 0.49 GACCCTTCGGTGACCRCAGGCTCCCTGCCAG

SGC31299 57 C 0.31 G 0.69 0.43 TGTCTAATTTTCCAASACTATGTTTAATGTA

SGC31303 117 C 0.56 T 0.44 0.49 GACTTCAGAGTAATAYGGTTTATGTCAGTTT

SGC31319 31 C 0.81 T 0.19 0.30 CCCACAATTTTGATTYGGTGGCTTCATAAGG

SGC31324 45 A 0.81 C 0.19 0.30 GAATAACTGATGTTCMCAATACCCCGACCCC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC31372 81 A 0.75 G 0.25 0.38 GACATCTAACATTAGRTAGCCTTCAGAATTG

SGC31485 84 C 0.25 G 0.75 0.38 TTTTCTCTATTCTCTSCAATTTGTTTAAAGA

SGC31490 131 C 0.88 T 0.13 0.22 TATATATATGGCTTTYCAATAACCACCTAAA

SGC31493 138 G 0.69 A 0.31 0.43 ACTTTGAAATGTAACRAATGGTACTACAACC

SGC31494 129 T 0.50 C 0.50 0.50 CCTCTGCTGCCATGGYGTGTCCCTCTCGGAA

SGC31500 103 C 0.81 T 0.19 0.30 TCTTTTGGACCAAACYTTTTTGTCTTTAGAG

SGC31534 159 A 0.75 T 0.25 0.38 AGGAATCTGGGAATTWGCCCTGGCCTGAAAG

SGC31566 72 T 0.63 G 0.38 0.47 TTTTGTTTATGGATCKGATAAAATCTAGATC

SGC31576 106 G 0.88 C 0.13 0.22 CCAACGATCATATCTSTATGCCTCATTTTAT

SGC31596 24 C 0.56 T 0.44 0.49 GTGACGTATGTAGAAYGCTTAGGGTGTCCTC

SGC31598 44 C 0.94 T 0.06 0.12 TGCTCTCATCACCAGYTAGAGCTTCTTCCCG

SGC31656 88 G 0.81 A 0.19 0.30 CGACTACCAGCTGATRAAATACCTGCAAAGT

SGC31729 128 G 0.88 A 0.13 0.22 CCATTTTAATAAGTGRTATGCTTTCTGAACA

SGC31748 19 A 0.31 C 0.69 0.43 GGAGCTCTGAGGAGCMCACCAAGGGACGTGT

SGC31767 41 T 0.13 C 0.88 0.22 TATTGAGTTATAATAYACATAAAAATCCACC

SGC31767 54 A 0.13 G 0.88 0.22 TATACATAAAAATCCRCCACTGTAAACAGTA

SGC31767 92 T 0.13 C 0.88 0.22 ATGGTTTTTACTCTAYTGTCAAAGCTGGGCA

SGC31772 74 C 0.38 T 0.63 0.47 CGGCACAGACAGAGTYTGGGAGCCATGGGGC

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC31777 118 C 0.88 G 0.13 0.22 GGAGATGCCCCATGTSTTGTGAGACTTAAAA

SGC31788 48 G 0.63 C 0.38 0.47 AAAGACAACAGAGGASAGCAGAGAATAATAT

SGC31914 100 A 0.94 G 0.06 0.12 TTTACATTCAAGGACRGCTTCCAGACAAGCC

SGC31986 61 T 0.44 G 0.56 0.49 TAAGAGGCATAATCTKAAACAAAATTCTTTC

SGC32030 51 A 0.38 G 0.63 0.47 GAACAATATTTTAGGRATTTGAAATTATTTC

SGC32039 69 G 0.94 C 0.06 0.12 GACTAGTTCAAGCAGSAGGTTAGACCAGTAA

SGC32060 115 T 0.13 G 0.88 0.22 CGGGAGTGCTGATTGKTCGGGTCCAAGATAA

SGC32109 78 T 0.88 C 0.13 0.22 TGCTATTCCTGCCATYACCGCATCCTTCATG

SGC32119 31 T 0.63 A 0.38 0.47 TGTTTCTTCTTTAAAWATGGTATAAAAATAA

SGC32190 27 C 0.88 T 0.13 0.22 CCAGGCTGGTCTCATYTCAGGCTCATGCGAT

SGC32204 91 T 0.63 C 0.38 0.47 CATTTTTCATCCTAAYTTACTGAAGCCATTT

SGC32206 40 A 0.25 C 0.75 0.38 TTAAGGGTATAGTTCMAGTGGCATTAAGTAC

SGC32206 41 A 0.38 G 0.63 0.47 TAAGGGTATAGTTCARGTGGCATTAAGTACA

SGC32299 108 T 0.94 A 0.06 0.12 ATTAATCTTTGCCTTWATGGTTTTGACAGTT

SGC32391 44 G 0.25 A 0.75 0.38 TTTCAATACTAAACARTGTAAACAATGCAAA

SGC32394 31 T 0.63 G 0.38 0.47 GTTTTGTTTTTTCCTKTATTGATGGGATTTA

SGC32407 51 C 0.88 T 0.13 0.22 TGTTCTCCAGTCTTGYAGGTTACATAAGCCA

SGC32411 98 T 0.81 C 0.19 0.30 TTCTCTCAAGTCCCTYTCATCCATACCACCA

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC32541 99 A 0.94 G 0.06 0.12 TTATTTTAATATTCCRGGATTTAATTTCTTC

SGC32577 91 T 0.13 G 0.88 0.22 GATGCCAATACTTCGKGCTTCCCAGAGTGCA

SGC32579 24 C 0.44 T 0.56 0.49 CCTAAAAGATCTTTTYTCCCCCAAGTCCTAA

SGC32586 101 C 0.63 G 0.38 0.47 CCTGCTCCGCCCTTCSGCCACCATCCATTCC

SGC32590 86 A 0.63 G 0.38 0.47 CCTGAGGTGATATGGRCCTTAAGTCCACGAT

SGC32609 72 T 0.94 C 0.06 0.12 ATTCCTAAAATCTATYACACTGAGAGGAAAA

SGC32612 63 G 0.69 T 0.31 0.43 TGAAACAGGGATGCCKTTCTCGGTACTATGT

SGC32620 86 A 0.63 G 0.38 0.47 GATTAGCGTGAGAGGRAAAAATGTGAAATGT

SGC32638 97 T 0.63 C 0.38 0.47 TTTCCAGTTGGTAAGYAGCAGGTGCCGAGGG

SGC32641 26 C 0.75 T 0.25 0.38 CGACGCCGGCGAGTTYGTGGACCTGTACGTG

SGC32650 83 A 0.69 C 0.31 0.43 CCTTGTTCAGATTTCMAAATAGTTGTAGCCT

SGC32816 79 C 0.75 T 0.25 0.38 TATATGTGCAGGGCCYGGGCGGGTGAAGGGT

SGC32859 78 A 0.13 G 0.88 0.22 TGGAACCTGAAACACRGACGCCTTTCTTCCA

SGC32871 39 C 0.94 T 0.06 0.12 TCATCCCAGATTATTYTGAAGTGGAAACCAC

SGC32871 128 C 0.75 T 0.25 0.38 AGACAGTGAGCTGTTYGAGCTGGATTATTGC

SGC32909 26 A 0.75 C 0.25 0.38 GGTAACCAGTTTTGTMACATTATTCAGAACT

SGC32909 95 C 0.44 G 0.56 0.49 GGAGAAAGCAGTGTGSTATAATGTCAACATC

SGC32942 92 G 0.44 C 0.56 0.49 GCGCCGGGCCTGCCCSGGACCCTGGTTTCCC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC32968 73 T 0.63 G 0.38 0.47 TTTGTTGTTGTTGTTKTTTTAATTATAAGAA

SGC32975 100 T 0.81 A 0.19 0.30 ACCTTCAAAAATTAAWTGTGACTTACGGAAA

SGC32978 58 T 0.88 C 0.13 0.22 CAGCTTGTATTACTTYACAAGTCAGACCTGT

SGC32986 114 A 0.88 G 0.13 0.22 TAGCAGCTTTTAGGGRTTATATCATGAGGTA

SGC32991 56 T 0.88 C 0.13 0.22 ACTGGATAAATAAAAYGTGGTACATGTACAC

SGC32993 38 C 0.44 T 0.56 0.49 TTTATATAAAACCTGYAGATGAATATTTTTT

SGC33004 53 A 0.63 T 0.38 0.47 ATTTTGGCTAATTTGWTAGTCTTACAAAGGC

SGC33092 96 T 0.75 C 0.25 0.38 CATTAAAAATGAACTYGGAATAAGAGCATAA

SGC33161 101 G 0.81 T 0.19 0.30 CATTTAAGAATGAAGKGGAAATGAAGGCAAT

SGC33169 109 C 0.88 T 0.13 0.22 CTCATCTGCTGGTGTYTTCCTCAGAGCTTTA

SGC33221 74 A 0.63 G 0.38 0.47 ACTACTCTTCCTTCARGACTATTTCATTCTG

SGC33235 82 G 0.94 A 0.06 0.12 CACATAGATCCCAGARTATTAAAGGGGCTGG

SGC33289 52 C 0.63 G 0.38 0.47 AGGTCACACTTGTCASCAGCAAGTATAAACA

SGC33301 95 A 0.50 G 0.50 0.50 ATTAACTGAGATTATRGGAAACGCACAGCAA

SGC33302 25 A 0.94 G 0.06 0.12 TTCTGGGCCTGTCAGRAAGTGACATCTTTTA

SGC33319 22 C 0.50 A 0.50 0.50 TCTCCAGGATTCCAGMCTCGTAGCTGATGTG

SGC33355 66 A 0.75 G 0.25 0.38 GCAGTGTCTGGAGACRGTTTTGATTGTCACA

SGC33366 45 C 0.06 G 0.94 0.12 ATGATTCAGCATTTASACTTTAAAAATTACC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC33368 69 T 0.75 C 0.25 0.38 GTCTAGGAGTAGAAAYGCACACAAGGAATAA

SGC33387 105 T 0.69 C 0.31 0.43 CAGTGTTGCCTGAGAYGATGCATGTGGCAGA

SGC33388 67 A 0.31 G 0.69 0.43 CATTGTCCACCGGGCRTTGAGAATACAATAT

SGC33424 104 C 0.94 T 0.06 0.12 TCTTCTAGGGCCACAYGGAGCAGAAGCAGCT

SGC33431 66 A 0.75 G 0.25 0.38 CCAAATAAAATGCACRTATTTAAAGTTTACA

SGC33436 44 C 0.75 T 0.25 0.38 TAGGTTTGTTCTTCCYAGCATATTCAGCTAT

SGC33475 101 C 0.44 G 0.56 0.49 ACTTTCTTGAACAAASTGATTACGAAAGTGA

SGC33492 25 A 0.75 G 0.25 0.38 CTGTAACCCGAGCCCRCAGTGACCGGGACTT

SGC33497 63 G 0.13 A 0.88 0.22 GCCTCACACAAGCATRATCAATCGCCACGAG

SGC33497 80 G 0.94 A 0.06 0.12 TCAATCGCCACGAGARACTGGATGCCAAAGA

SGC33499 23 A 0.56 G 0.44 0.49 TCCATGTGAACATATRACCTATTCATAAAGT

SGC33533 58 C 0.75 T 0.25 0.38 AATACGAACAGTGCAYGCTGATGGCCTGCAG

SGC33533 102 G 0.88 A 0.13 0.22 TTGGCTCTCTGGACGRTTCATTCTACATGGC

SGC33565 89 G 0.63 C 0.38 0.47 CAGAAAAGGCCGCTCSGGGTTTTCTGAACCC

SGC33567 96 C 0.75 T 0.25 0.38 CCTAAGTAGTCTCTCYAAAGAGCCATCCCTG

SGC33570 109 C 0.56 A 0.44 0.49 TATAGCCAAGGGACTMGGAATTTTGGCTGCT

SGC33582 58 T 0.50 C 0.50 0.50 CAGGGCAACATAGGAYTGTGACAGCACCACT

SGC33603 53 C 0.31 T 0.69 0.43 CTAGAGGAGAGATTAYAATGAACGTAAATAA

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag" Allele" (P)" Allele" (Q)"

SGC33608 83 A 0.75 G 0.25 0.38 TGGTTCCTCCAGGGARTTGGCCCCGAAGCTG

SGC33610 28 G 0.69 A 0.31 0.43 TGGCTTTCAAAATCARTACAGACAGATAAGA

SGC33623 95 C 0.38 A 0.63 0.47 GCCGAGGTCACTGCTMTACAAAGATTAAAGA

SGC33642 24 C 0.88 G 0.13 0.22 GTGAAGGGACAGGAGSGTAAACACAGTCCAT

SGC33691 32 C 0.94 G 0.06 0.12 GTTCCAGGTTTTTTTSTGAACAAATGATCCT

SGC33691 119 T 0.63 C 0.38 0.47 CAGTCACCTAAGATAYCGAGTGGCAAGTCTT

SGC33707 54 G 0.75 A 0.25 0.38 ATGGTAAAACAGCAGRAAATGGAATTATAGC

SGC33710 59 A 0.19 G 0.81 0.30 AGAAATATCTAGTTGRGTAGAGGAAGGCACT

SGC33712 28 A 0.69 G 0.31 0.43 ATGACACTGCCAACARTCACAGATTTGCATA

SGC33724 45 T 0.69 C 0.31 0.43 GAGTCACAGTTTCATYTGGGAGTCCCTGTGC

SGC33724 52 T 0.44 C 0.56 0.49 AGTTTCATTTGGGAGYCCCTGTGCAGCCCTT

SGC33731 49 G 0.19 A 0.81 0.30 TGTCCCAGTGCCACARTGGTCTAGCCTCATG

SGC33736 62 C 0.44 G 0.56 0.49 TTCAGTTGACAGATTSTCTCCTTACCTAACT

SGC33754 55 C 0.25 T 0.75 0.38 TTGACTCAAGGGCATYGTAATAGGTTTCCAT

SGC33754 69 A 0.06 G 0.94 0.12 TCGTAATAGGTTTCCRTACTGCAGAAGAAGG

SGC33764 71 C 0.25 T 0.75 0.38 GGAAAACAGGAAATCYATCCTTCAAGCATTA

SGC33768 41 A 0.13 G 0.88 0.22 AGGCATGAGGAGCTGRTTATGCAGATATACT

SGC33773 38 G 0.38 A 0.63 0.47 ACAACTTGCAAGCACRGGGAGAAAACCTAGG

SGC33835 32 C 0.06 T 0.94 0.12 TTTGTCCATTGTTGAYTGTGAATAATTGGCA

SGC33887 118 T 0.63 C 0.38 0.47 TCACATAGGCAGTTGYACACCCAGCTGACAA

SGC33917 72 T 0.50 C 0.50 0.50 GACATGTGGTGGCTGYGAGGGAGAAGGACCC

SGC33945 21 C 0.75 T 0.25 0.38 TTGCTTAGCCAGCTTYATCAGTGGTGCCCTA

SGC33952 102 C 0.75 G 0.25 0.38 CATAAATTATCAAATSTGCGCCCAGTAATCT

SGC33970 76 C 0.88 G 0.13 0.22 CCCTACTTAGACCCTSGCACACAAAGGTTGA

SGC33989 31 T 0.88 A 0.13 0.22 AGTCCCTAGGTGTGTWTGAAACAATCTGGGT

SGC33991 93 A 0.75 G 0.25 0.38 AAATCACAGTACTGGRATCAGGTGAAATTTG

SGC34004 90 T 0.75 C 0.25 0.38 AGCAAACCAATAAAAYCATATATCTTGAGGG

SGC34009 46 G 0.50 A 0.50 0.50 TAAGACAGTGCTCACRTGGCCTGAATGTTGG

SGC34014 75 T 0.25 A 0.75 0.38 GGTACCAATATCAATWCAGTTTTCAAAGCCA

SGC34014 98 T 0.88 C 0.13 0.22 CAAAGCCATTTGCAGYACTCTTCAGATGGGT

SGC34016 44 T 0.94 C 0.06 0.12 AACGGTTTGTAGTTTYGCTTACCCGCAGTGC

SGC34029 53 A 0.56 G 0.44 0.49 CAGATCTGTTTTCAGRAAGAGGGCCTACTTT

SGC34033 86 C 0.75 T 0.25 0.38 TTTTGACCTATCTCAYCAAGCGAGAGGGAGG

SGC34033 107 G 0.75 A 0.25 0.38 GAGAGGGAGGCAAGCRGAGGGATGGTTTATC

SGC34037 68 A 0.63 G 0.38 0.47 CTGATGGAAGCATCARTGATGGATTTGGCTT

SGC34039 63 T 0.88 C 0.13 0.22 CAGCTTGTGTTGATGYCTACAAAGAAGTCAG

SGC34088 64 T 0.88 C 0.13 0.22 CAAAGCTGAAACTAAYGAGTGAGCATAGCAA

SGC34119 25 T 0.94 C 0.06 0.12 ATGGAAAGAGTGACAYCCTTGTCCTGTTCTG

SGC34142 49 A 0.38 G 0.63 0.47 GAAAACTGATACACCRGTTACTACTTACTCT

SGC34145 80 G 0.50 A 0.50 0.50 TACTAGGTGCTGGGARTGTGACAGTGAGCAA

SGC34158 26 A 0.44 G 0.56 0.49 AATGACAAAGCCCAARAGAACAGAGGATCAA

SGC34223 106 T 0.63 C 0.38 0.47 TTTAGCGTAAATACCYGAATAACCCATAGTT

SGC34226 73 G 0.88 A 0.13 0.22 CAGGCATAAGCAGCCRTGCCTGACCCACATT

SGC34248 25 T 0.81 C 0.19 0.30 AAAGTAAGCAGCCGGYTGGTCCCTGGATTGA

SGC34278 33 G 0.81 A 0.19 0.30 GACCTGCTCCTAAAARCTTTCTCCTCCTCCT

SGC34351 51 G 0.25 A 0.75 0.38 CTGTGAACTATGAACRTCTCAGCCTAGAAGG

SGC34363 28 T 0.13 C 0.88 0.22 CTACCAGAACTCATGYGATAGCGCTTTCTTT

SGC34377 78 A 0.31 T 0.69 0.43 GGAAACTTACAATCAWGGTAGAAGGCAAAAG

SGC34392 56 T 0.50 G 0.50 0.50 GTTTTATATCACTTAKTTATCTCAACAATCT

SGC34411 50 G 0.75 A 0.25 0.38 AGCTCTCAGGACTGGRGCTAGGGTTTAAGGA

SGC34413 59 C 0.75 G 0.25 0.38 AAAGTTCAGTAGAGASAGGTGTTTTGAATGT

SGC34485 77 C 0.88 T 0.13 0.22 AAATGATCATTTAACYTCTTTGAACTACAGC

SGC34486 75 C 0.81 G 0.19 0.30 CTTAGAGAAGTTTAASGCACATAGTATTATT

SGC34488 33 G 0.88 T 0.13 0.22 CATGACTACCAACGCKGGCCCCTTGCACCCA

Fragment Position "Ref "Freq "Alt "Freq "H" "Sequence Tag" Allele" (P)" Allele" (Q)"

SGC34489 27 T 0.94 C 0.06 0.12 CTCCAAATCCTAAAAYGTGTGTCTTCAAAGA

SGC34498 126 A 0.75 C 0.25 0.38 TACACACTGAGCAACMAAACAAAGGTGTTGA

SGC34531 90 A 0.44 G 0.56 0.49 TAACATCGTCTATAGRACCATTTCCCGTCTC

SGC34575 126 C 0.94 G 0.06 0.12 ATTTTGATGCAGTTTSGTTAGGGAATTAAGA

SGC34640 97 T 0.67 A 0.33 0.44 GCTGTGGGGAACCTCWGGTGCCTTACAACTC

SGC34662 25 G 0.50 A 0.50 0.50 GGAAAAAATGGTGGCRTGCCTCTAAAACCTG

SGC34671 104 A 0.83 G 0.17 0.28 CAGGATGTTCCCTGARGTATTCAGGAATTCT

SGC34681 82 G 0.08 A 0.92 0.15 TGGGGAGTCTATGTTRTGCTTTCTGGTGGCC

SGC34681 93 T 0.92 G 0.08 0.15 TGTTGTGCTTTCTGGKGGCCTTAAAAGAAAC

SGC34724 93 G 0.42 T 0.58 0.49 ATAAAAGAGGTTCTCKGCCTTTCCAGCGTTG

SGC34725 33 C 0.83 T 0.17 0.28 TGTAGGCATTTAATGYTATAAATTTCCTGCT

SGC34755 32 C 0.83 T 0.17 0.28 TTAGGCAAATGGAAAYAGACTTACTGTATGG

SGC34764 51 C 0.83 G 0.17 0.28 CCCACAAAGGCTCCASATGTTAAAACGTTTC

SGC34765 89 C 0.92 A 0.08 0.15 CCCATGAAACCAAGAMCTTGTCCTCATGATA

SGC34830 62 C 0.88 T 0.13 0.22 TACTGATTGACAATGYATATTAGCCAGGTAA

SGC34846 93 C 0.25 T 0.75 0.38 CAGCCATGGCCCCTGYGCTGATGGAGCTTGT

SGC34858 89 C 0.25 G 0.75 0.38 GTCTGGGGATTCCTASAGGGGACATATCACA

SGC34906 103 A 0.31 T 0.69 0.43 ATTCAAGCAACAATTWTCTTTTATGTTCCTA

SGC34924 88 T 0.56 C 0.44 0.49 TGCTAGGATTACAGGYGTGAGCCACCACACC

SGC34951 37 T 0.94 C 0.06 0.12 AGGGAAACTAAGCTCYTCAAAATAACTGAAA

SGC34953 42 A 0.75 G 0.25 0.38 CACTACGCATGCACARATAAAGTCACATCAA

SGC34961 81 A 0.94 C 0.06 0.12 TGAGCTGGTGGAAAAMGGACTTGGAGACAGC

SGC34964 31 C 0.94 A 0.06 0.12 CATAGTGCCTCTAGTMACCTATGAGGCACTA

SGC34974 68 A 0.94 G 0.06 0.12 TGTAATGCACACCCARTCTGTACTCCCACAA

SGC34978 105 T 0.56 G 0.44 0.49 TATATTTTGAAAGTCKCAGGAGAAAAAATGG

SGC34982 93 G 0.63 T 0.38 0.47 ATGTCACTCTAGGAAKAGTAAACAGGTGTTA

SGC34985 41 T 0.69 C 0.31 0.43 TTCAATTAATAGTAGYTGAGCGCTGGGGGCT

SGC34985 101 G 0.88 C 0.13 0.22 GTGCTGTGTCCTGCASGCTGTCCTCAGGCAA

SGC34990 63 T 0.56 G 0.44 0.49 TCAATTCGTGAAAACKAACATGCCTCAAAAA

SGC34994 90 A 0.88 G 0.13 0.22 ATAGTAGGAGTATCTRCCCTGCCCTGCTAGA

SGC35006 45 C 0.56 T 0.44 0.49 ATCCTCCTCAAACTTYAAGGGTGAAAAGCAT

SGC35020 46 G 0.06 A 0.94 0.12 AAATATTAAACCTCTRCTTCTCAGGAGTGAC

SGC35053 34 A 0.25 G 0.75 0.38 AGTCATTTATTTACCRGTCATGAATTCATTA

SGC35081 57 C 0.56 T 0.44 0.49 TTTTCATGTCACTTAYCGCATGGAAGAACGC

SGC35145 100 T 0.38 C 0.63 0.47 GTCATCCTGACTGACYGTCCCTGCAGTGCCC

SGC35186 90 T 0.63 C 0.38 0.47 TACACAAATGCTATGYAAACAAGTTACTGAA

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC35222 70 G 0.88 C 0.13 0.22 GGAAGGAAAGTATGCSGTGTTTTAGGGAGAG

SGC35233 21 T 0.50 C 0.50 0.50 CATGCGTGTGACCTCYACAGCTACCTCTTCT

SGC35238 159 G 0.19 A 0.81 0.30 GCTCTTCATTCTCACRGGCCCGCAACCCCTC

SGC35244 81 A 0.56 G 0.44 0.49 GACCTCCTGTGACCCRTGAATGTGCCTCCAA

SGC35245 166 C 0.88 T 0.13 0.22 TAATACGTACTTTAGYTGGAATTATTCTATG

SGC35252 39 T 0.31 C 0.69 0.43 TCAGGAACACCCCCAYGACATTGCATTTGGG

SGC35267 134 T 0.63 C 0.38 0.47 TTATCCAACTCTCGAYTTTTCCTTGGTCTCC

SGC35276 39 T 0.88 C 0.13 0.22 ATCTGTATTGACTAAYACACCAGTCCACACT

SGC35282 45 C 0.44 G 0.56 0.49 TATCCCTTTTCTCCTSCAAATGTTTCTCCTC

SGC35282 157 A 0.31 G 0.69 0.43 TTTTTTCTTTTCTCARGTGTTACCTACTAAG

SGC35282 173 A 0.50 G 0.50 0.50 GTGTTACCTACTAAGRGATGCCTGGAGTAAG

SGC35285 63 T 0.63 C 0.38 0.47 TCATGTGAAAACTACYCCAGTGGCTGACTGA

SGC35326 34 G 0.81 A 0.19 0.30 TCAGGCTGACGGGGARGAACCACTGCACCAC

SGC35336 36 T 0.88 C 0.13 0.22 CCTTTAGGGCTACAGYCTCTTGTCCTGGACC

SGC35345 137 G 0.50 C 0.50 0.50 CAGCGTCCCCCACCCSCGTCGTGGTGTAGTC

SGC35346 133 A 0.75 G 0.25 0.38 TGACTGCATGAATGCRTGTGCGTGCAAGCAT

SGC35357 123 T 0.94 G 0.06 0.12 TTGTATTTTGTATATKCGCCTGAAGATCATC

SGC35364 21 A 0.56 G 0.44 0.49 CATCCTGATGCCCCARGTTATCCACAGCCTC

Fragment Position "Ref "Freq "Alt "Freq H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC35364 85 A 0.88 G 0.13 0.22 CATTTTTCCCTGTAARTTCTCCAACTGATCC

SGC35370 162 T 0.25 C 0.75 0.38 GAACAGCCAAGAGATYTTACCGTGGTCTTAC

SGC35384 58 T 0.81 C 0.19 0.30 GTAGTTCTAGAACTTYAGAAGCTCCATCTTT

SGC35405 114 T 0.75 G 0.25 0.38 TCCCCGACAGCAAAAKGTTTCCTTTCTGAGG

SGC35413 62 T 0.88 G 0.13 0.22 TTGTATATAAGATAAKTCATACTGGAGAAAA

SGC35416 143 C 0.88 G 0.13 0.22 AAAATTGCAAAAAGAMAAGTATGACTTTTAT

SGC35416 164 C 0.75 A 0.25 0.38 AACCTCAACCACATCYTATCCTCCACCCCAC

SGC35419 25 T 0.31 C 0.69 0.43 CCCTTCCTGGAGACTRAACCTGGTGCTCAGG

SGC35432 147 G 0.19 A 0.81 0.30 TATGTTATTTGCTCTRATACAAAAATTCTAA

SGC35438 99 A 0.88 G 0.13 0.22 GGGAGGGGGCGTTTCRCTTTCCTTCTTCTTG

SGC35461 82 G 0.88 A 0.13 0.22 GGGAGGGGGCGTTTCRCTTTCCTTCTTCTTG

SGC35464 128 T 0.69 G 0.31 0.43 CCGCAAGATGGGGCCKGGGCATGCGCAGGAG

SGC35477 179 G 0.50 T 0.50 0.50 ACAAGATGGAATTTAKCAAACCCTAGCCTTG

SGC35498 173 G 0.75 A 0.25 0.38 ACCACAAATCTGAACRTGCCTCTCCCTTGCT

SGC35499 68 C 0.50 T 0.50 0.50 CTTAGGGCATCGCTCYTCCTCACGCCACAAA

SGC35499 76 G 0.75 A 0.25 0.38 ATCGCTCCTCCTCACRCCACAAATCTGGTGC

SGC35527 82 T 0.38 C 0.63 0.47 GGGAAAGTCTGGTCCYACATCTGCCCGCCCT

SGC35529 54 A 0.63 G 0.38 0.47 GGCCCTGAGCGTCCTRCCCCGAATTCACGAG

SGC35531 57 G 0.06 C 0.94 0.12 CACAACCACCTTGACSAATGCTTGCCAAGCT SGC35537 187 T 0.81 C 0.19 0.30 GCAGTCTGGTCCATGYTGGTCTCATACCTCA SGC35543 78 C 0.81 T 0.19 0.30 CCTCCAGACCGCAGGYTCCCCCAGCCTCAGG SGC35548 61 A 0.06 G 0.94 0.12 GGTGTTGACACACCARTTTTGAGTGTACTGT SGC35566 70 G 0.88 A 0.13 0.22 TGCAACCAAACAGCCRTCATCAAACCCCTCA SGC35566 78 A 0.44 G 0.56 0.49 AACAGCCGTCATCAARCCCCTCACTAAAAGT SGC35569 28 A 0.56 G 0.44 0.49 TCCCAGGCCCCAGGCRTCTTTCCTGCCCTGC SGC35569 99 G 0.63 C 0.38 0.47 TCAGCTACTTCTCCTSCACTTTGAAAGACCC SGC35579 29 C 0.31 T 0.69 0.43 AATTAGCCCTAAATGYGGGTAATATTTTTCC SGC35580 64 T 0.81 C 0.19 0.30 AATGCATTTGAGCTGYCCCAGGCTCTGTCTC SGC35587 118 A 0.25 C 0.75 0.38 ACATTCACAAAGAAAMGTTGCGAAAATTGCG SGC35587 148 C 0.63 T 0.38 0.47 GAAATCTGTTGTGCAYGCTCAAATGAAAACG SGC35590 191 A 0.56 C 0.44 0.49 TGCAGCTTAAAGAGCMCAGGTTCCAGTACTG SGC35597 69 T 0.19 C 0.81 0.30 CACCTTCCAAGGCCCYATCCATTAGTTTCCA SGC35598 24 A 0.00 G 1.00 0.00 TGTCTTCTCTCCCACRTGCACAGCTTCCTGA SGC35601 113 T 0.56 C 0.44 0.49 CTCCCCATGTGCCTGYGCCAAGAGACAGACA SGC35615 52 G 0.63 A 0.38 0.47 TATTGTACCAGAACTRTTTATTTCACCCCAT SGC35626 106 A 0.69 T 0.31 0.43 CATGTGGTTTTAAAAWATCCATAAGGGAAGG

Fragment Position "Ref "Freq "Alt "Freq Η" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC35638 20 C 0.19 G 0.81 0.30 ATGAGGCCCATTCTTSGCTCTGTGTTTGAAG

SGC35645 122 T 0.81 C 0.19 0.30 GCGATGACACCACACYTTGTTTGGACATTTA

SGC35655 101 C 0.25 T 0.75 0.38 ATTTTTCCTGTTCCAYGAAGAGGACTTTTTG

SGC35659 150 G 0.19 A 0.81 0.30 TCTTTTCTCCCAAGCRAAACCAAATGCGCCC

SGC35665 34 A 0.88 G 0.13 0.22 TCAGGAGTCATTAGCRGTGATGATTTTGGGA

SGC35665 89 A 0.31 G 0.69 0.43 TTCCCACGTTAGCCARTTGTTCTTGATGAAT

SGC35671 111 T 0.25 C 0.75 0.38 GGTTTACTTTCAGAAYGAAGAACTTATTCAG

SGC35678 34 C 0.88 T 0.13 0.22 TGACTCTGCTTCCTGYACTGACCCAGAGCCT

SGC35687 70 T 0.56 A 0.44 0.49 ATCTCTAAATAAGATWACATTCTGGGGTACT

SGC35825 57 C 0.63 T 0.38 0.47 TAGTAATAAATTACAYGAGATATTCACACTT

SGC35842 98 A 0.94 G 0.06 0.12 ATCCATTATTTACAGRAAATGTGGAAAAGAT

SGC35914 59 T 0.88 C 0.13 0.22 TTATTTATGAGCCCCYGAGGACCAGACATGT

SGC35927 71 C 0.06 T 0.94 0.12 TTCAGTATCATTATGYTGTAGATTTCAGATG

SGC35928 25 T 0.50 C 0.50 0.50 TTATCAAAATGGTTAYAGTTTTCAATTAAAA

SGC35946 45 A 0.25 G 0.75 0.38 AATTTTTCTCAACTTRCATTTAAAAATGTAT

SGC35965 25 A 0.50 C 0.50 0.50 GACATACATATCTCAMGTAGAATTAGCTATA

SGC35978 36 A 0.88 G 0.13 0.22 ACTTTTTTATAAAGARTAAGTTGACTGAAAA

SGC35978 45 C 0.50 T 0.50 0.50 TAAAGAATAAGTTGAYTGAAAAGCAGTTTTA

Fragment Position "Ref "Freq "Alt "Freq "H" "Sequence Tag"

Allele" (P)" Allele" (Q)"

SGC36020 26 T 0.56 C 0.44 0.49 ACAAGACAATTGCATYTAACATTGTTATAAA

SGC36047 31 T 0.63 A 0.38 0.47 AGACGGACATAAAAAWTATACAACAAAAAAC

Analysis of Polymorphisms

A. Preparation of Samples

Polymorphisms are detected in a target nucleic acid from an individual being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed. For example, if the target nucleic acid is a cytochrome P450, the liver is a suitable source.

Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally PCR Technology:

Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992) ; PCR Protocols : A Guide to Methods and Applications (eds. Innis, et al . , Academic Press, San Diego, CA, 1990); Mattila et al . , Nucleic Acids Res. 19, 4967 (1991); Eckert et al . , PCR Methods and

Applications 1, 17 (1991); PCR (eds. McPherson et al . , IRL Press, Oxford); and U.S. Patent 4,683,202 (each of which is incorporated by reference for all purposes) .

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al . , Science 241, 1077 (1988), transcription amplification (Kwoh et al . , Proc. Natl . Acad. Sci . USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al . , Proc. iVat. Acad. Sci . USA, 87, 1874 (1990) ) and nucleic acid based sequence amplification (NASBA) . The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively. B . Detection of Polymorphisms in Target DNA

There are two distinct types of analysis depending whether a polymorphism in question has already been characterized. The first type of analysis is sometimes referred to as de novo characterization. This analysis compares target sequences in different individuals to identify points of variation, i.e., polymorphic sites. By analyzing a groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals, patterns characteristic of the most common alleles/haplotypes of the locus can be identified, and the frequencies of such populations in the population determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender. The de novo identification of the polymorphisms of the invention is described in the Examples section. The second type of analysis is determining which form(s) of a characterized polymorphism are present in individuals under test. There are a variety of suitable procedures, which are discussed in turn.

1. Allele-Specific Probes

The design and use of allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al . , Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.

2. Tiling Arrays The polymorphisms can also be identified by hybridization to nucleic acid arrays, some example of which are described by WO 95/11995 (incorporated by reference in its entirety for all purposes) . One form of such arrays is described in the Examples section in connection with de novo identification of polymorphisms. The same array or a different array can be used for analysis of characterized polymorphisms. WO 95/11995 also describes subarrays that are optimized for detection of a variant forms of a precharacterized polymorphism. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same principles as described in the Examples except that the probes exhibit complementarily to the second reference sequence. The inclusion of a second group (or further groups) can be particular useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (i.e., two or more mutations within 9 to 21 bases) .

3. Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarily. See Gibbs, Nucleic Acid Res . 17,

2427-2448 (1989) . This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present . A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarily to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3 ' - most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer. See, e . g. , WO 93/22456. . Direct-Sequencing

The direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al . , Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al . , Recombinant DNA Laboratory Manual , (Acad. Press, 1988)).

5. Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed. , PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992) , Chapter 7.

6. Single-Strand Conformation Polymorphism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al . , Proc . Nat . Acad. Sci . 86, 2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target sequences .

III. Methods of Use

After determining polymorphic form(s) present in an individual at one or more polymorphic sites, this information can be used in a number of methods . A. Forensics

Determination of which polymorphic forms occupy a set of polymorphic sites in an individual identifies a set of polymorphic forms that distinguishes the individual. See generally National Research Council, The Evaluation of Forensic DNA Evidence (Eds. Pollard et al . , National Academy Press, DC, 1996) . The more sites that are analyzed the lower the probability that the set of polymorphic forms in one individual is the same as that in an unrelated individual. Preferably, if multiple sites are analyzed, the sites are unlinked. Thus, polymorphisms of the invention are often used in conjunction with polymorphisms in distal genes. Preferred polymorphisms for use in forensics are diallelic because the population frequencies of two polymorphic forms can usually be determined with greater accuracy than those of multiple polymorphic forms at multi-allelic loci.

The capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymorphic forms occupying selected polymorphic sites is the same in the suspect and the sample. If the set of polymorphic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies of the polymorphic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals) , one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance . p(ID) is the probability that two random individuals have the same polymorphic or allelic form at a given polymorphic site. In diallelic loci, four genotypes are possible: AA, AB, BA, and BB . If alleles A and B occur in a haploid genome of the organism with frequencies x and y, the probability of each genotype in a diploid organism are (see WO

95/12607) : Homozygote: p (AA) = x²

Homozygote: p(BB)= y² = (1-x)²

Single Heterozygote : p(AB)= p(BA)= xy = x(l-x)

Both Heterozygotes : p (AB+BA) = 2xy = 2x(l-x)

The probability of identity at one locus (i.e, the probability that two individuals, picked at random from a population will have identical polymorphic forms at a given locus) is given by the equation: p(ID) = (x²)² + (2xy)² + (y²)².

These calculations can be extended for any number of polymorphic forms at a given locus. For example, the probability of identity p(ID) for a 3 -allele system where the alleles have the frequencies in the population of x, y and z, respectively, is equal to the sum of the squares of the genotype frequencies : p(ID) = x⁴ + (2xy)² + (2yz)² + (2xz)² + z⁴ + y⁴

In a locus of n alleles, the appropriate binomial expansion is used to calculate p(ID) and p(exc) .

The cumulative probability of identity (cum p(ID)) for each of multiple unlinked loci is determined by multiplying the probabilities provided by each locus. cum p(ID) = p(IDl)p(ID2)p(ID3) .... p(IDn)

The cumulative probability of non- identity for n loci (i.e. the probability that two random individuals will be different at 1 or more loci) is given by the equation: cum p(nonΙD) = 1-cum p(ID) .

If several polymorphic loci are tested, the cumulative probability of non-identity for random individuals becomes very high (e.g., one billion to one) . Such probabilities can be taken into account together with other evidence in determining the guilt or innocence of the suspect. B. Paternity Testing

The object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father. Paternity testing can be performed by analyzing sets of polymorphisms in the putative father and the child.

If the set of polymorphisms in the child attributable to the father does not match the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymorphisms in the child attributable to the father does match the set of polymorphisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match.

The probability of parentage exclusion (representing the probability that a random male will have a polymorphic form at a given polymorphic site that makes him incompatible as the father) is given by the equation (see WO 95/12607) : p(exc) = xy(l-xy) where x and y are the population frequencies of alleles A and B of a diallelic polymorphic site.

(At a triallelic site p(exc) = xy(l-xy) + yz(l- yz) + xz(l-xz)+ 3xyz (1-xyz) ) ) , where x, y and z and the respective population frequencies of alleles A, B and C) . The probability of non-exclusion is p(non-exc) = l-p(exc)

The cumulative probability of non-exclusion (representing the value obtained when n loci are used) is thus: cum p(non-exc) = p (non-excl) p (non-exc2) p (non-exc3) .... p (non-excn) The cumulative probability of exclusion for n loci (representing the probability that a random male will be excluded) cum p(exc) = 1 - cum p(non-exc) . If several polymorphic loci are included in the analysis, the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymorphic marker set matches the child's polymorphic marker set attributable to his/her father.

C. Correlation of Polymorphisms with Phenotvpic Traits

The polymorphisms of the invention may contribute to the phenotype of an organism in different ways. Some polymorphisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymorphisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymorphism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymorphisms in different genes. Further, some polymorphisms predispose an individual to a distinct mutation that is causally related to a certain phenotype.

Phenotypic traits include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent porphyria) . Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin- dependent and non-independent) , systemic lupus erythematosus and Graves disease. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus. Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity) , strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.

Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymorphic markers sets. To perform such analysis, the presence or absence of a set of polymorphisms (i.e. a polymorphic set) is determined for a set of the individuals, some of whom exhibit a particular trait, and some of which exhibit lack of the trait. The alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation can be performed by standard statistical methods such as a K- squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymorphism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymorphism A and allele Bl at polymorphism B correlates with increased milk production of a farm animal .

Such correlations can be exploited in several ways. In the case of a strong correlation between a set of one or more polymorphic forms and a disease for which treatment is available, detection of the polymorphic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient. Detection of a polymorphic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymorphism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymorphic set and human disease, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles. Identification of a polymorphic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.

For animals and plants, correlations between characteristics and phenotype are useful for breeding for desired characteristics. For example, Beitz et al . , US 5,292,639 discuss use of bovine mitochondrial polymorphisms in a breeding program to improve milk production in cows. To evaluate the effect of mtDNA D-loop sequence polymorphism on milk production, each cow was assigned a value of 1 if variant or 0 if wildtype with respect to a prototypical mitochondrial DNA sequence at each of 17 locations considered. Each production trait was analyzed individually with the following animal model : ^Yijkpn= M ^{+ YS}i ^{+ P}j ^{+ X}k ⁺ 01 ^{+ • • •} 017 ^{+ PE}n ^{+ a}n ^+ep where Yi_jknp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record; μ is an overall mean; YS.^ is the effect common to all cows calving in year-season; X_k is the effect common to cows in either the high or average selection line; β₁ to β₁₇ are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms; PE_n is permanent environmental effect common to all records of cow n; a_n is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect; and e_p is a random residual. It was found that eleven of seventeen polymorphisms tested influenced at least one production trait . Bovines having the best polymorphic forms for milk production at these eleven loci are used as parents for breeding the next generation of the herd.

D. Genetic Mapping of Phenotypic Traits

The previous section concerns identifying correlations between phenotypic traits and polymorphisms that directly or indirectly contribute to those traits. The present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymorphic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it. Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al . , Proc. Natl . Acad. Sci . (USA) 83, 7353-7357 (1986); Lander et al., Proc. Natl . Acad. Sci . (USA) 84, 2363-2367 (1987); Donis- Keller et al . , Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 185-199 (1989)). Genes localized by linkage can be cloned by a process known as directional cloning. See Wainwright, Med. J. Australia 159, 170-174 (1993) ; Collins, Nature Genetics 1, 3-6 (1992) (each of which is incorporated by reference in its entirety for all purposes) .

Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymorphic markers. The distribution of polymorphic markers in an informative meiosis is then analyzed to determine which polymorphic markers co-segregate with a phenotypic trait. See, e . g. , Kerem et al . , Science 245, 1073- 1080 (1989); Monaco et al . , Nature 316, 842 (1985); Yamoka et al., Neurology 40, 222-226 (1990); Rossiter et al . , FASEB Journal 5, 21-27 (1991) . Linkage is analyzed by calculation of LOD (log of the odds) values. A lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction θ , versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome" in The Human Genome (BIOS Scientific Publishers Ltd, Oxford) , Chapter 4) . A series of likelihood ratios are calculated at various recombination fractions ( θ ) , ranging from θ = 0.0 (coincident loci) to θ = 0.50 (unlinked) . Thus, the likelihood at a given value of θ is: probability of data if loci linked at θ to probability of data if loci unlinked. The computed likelihoods are usually expressed as the log₁₀ of this ratio (i.e., a lod score) .

For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of θ

(e.g., LIPED, MLINK (Lathrop, Proc. Nat . Acad. Sci . (USA) 81, 3443-3446 (1984)). For any particular lod score, a recombination fraction may be determined from mathematical tables. See Smith et al . , Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith,

Ann . Hum. Genet . 32, 127-150 (1968) . The value of θ at which the lod score is the highest is considered to be the best estimate of the recombination fraction.

Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of 0) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked. Similarly, by convention, a negative lod score of -2 or less is taken as definitive evidence against linkage of the two loci being compared. Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non- excluded chromosomal locations.

IV. Modified Polvpeptides and Gene Sequences

The invention further provides variant forms of nucleic acids and corresponding proteins. The nucleic acids comprise one of the sequences described in Table 1, column 8, in which the polymorphic position is occupied by one of the alternative bases for that position. Some nucleic acid encode full-length variant forms of proteins. Similarly, variant proteins have the prototypical amino acid sequences of encoded by nucleic acid sequence shown in Table 1, column 8, (read so as to be in-frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymorphic positions shown in the Table. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in the Table.

Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host. The selection of an appropriate promoter, for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.

The means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra . A wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli , yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e . g. , mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide.

Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.

The protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1987) ; and Deutscher (ed) , Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990) . If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.

The invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al . , "Manipulating the Mouse Embryo, A Laboratory Manual, " Cold Spring Harbor Laboratory. Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989) . The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems .

In addition to substantially full-length polypeptides expressed by variant genes, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures. Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Anti-odies, A Laboratory Manual , Cold Spring Harbor Press, New York (1988) ; Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986) . Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

V. Kits

The invention further provides kits comprising at least one allele-specific oligonucleotide as described above. Often, the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymorphism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymorphisms shown in Table 1. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin) , and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kit also contains instructions for carrying out the methods.

VI . Computer Systems For Storing Polymorphism Data

Fig. 1A depicts a block diagram of a computer system 10 suitable for implementing the present invention. Computer system 10 includes a bus 12 which interconnects major subsystems such as a central processor 14, a system memory 16 (typically RAM), an input/output (I/O) controller 18, an external device such as a display screen 24 via a display adapter 26, serial ports 28 and 30, a keyboard 32, a fixed disk drive 34 via a storage interface 35 and a floppy disk drive 36 operative to receive a floppy disk 38, and a CD-ROM (or DVD-ROM) device 40 operative to receive a CD-ROM 42. Many other devices can be connected such as a user pointing device, e.g., a mouse 44 connected via serial port 28 and a network interface 46 connected via serial port 30.

Many other devices or subsystems (not shown) may be connected in a similar manner. Also, it is not necessary for all of the devices shown in Fig. 1A to be present to practice the present invention, as discussed below. The devices and subsystems may be interconnected in different ways from that shown in Fig. 1A. The operation of a computer system such as that shown in Fig. 1A is well known. Databases storing polymorphism information according to the present invention can be stored, e.g., in system memory 16 or on storage media such as fixed disk 34, floppy disk 38, or CD-ROM 42. An application program to access such databases can be operably disposed in system memory 16 or sorted on storage media such as fixed disk 34, floppy disk 38, or CD-ROM 42.

Fig. IB depicts the interconnection of computer system 10 to remote computers 48, 50, and 52. Fig. IB depicts a network 54 interconnecting remote servers 48, 50, and 52. Network interface 46 provides the connection from client computer system 10 to network 54. Network 54 can be, e.g., the Internet . Protocols for exchanging data via the Internet and other networks are well known. Information identifying the polymorphisms described herein can be transmitted across network 54 embedded in signals capable of traversing the physical media employed by network 54. Information identifying polymorphisms shown in Table 1 is represented in records, which optionally, are subdivided into fields. Each record stores information relating to a different polymorphisms in Table 1. Collectively, the records can store information relating to all of the polymorphisms in Table 1, or any subset thereof, such as 5, 10, 50, or 100 polymorphisms from Table 1. In some databases, the information identifies a base occupying a polymorphic position and the location of the polymorphic position. The base can be represented as a single letter code (i.e., A, C, G or T/U) present in a polymorphic form other than that in the reference allele. Alternatively, the base occupying a polymorphic site can be represented in IUPAC ambiguity code as shown in Table 1. The location of a polymorphic site can be identified as its position within one of the sequences shown in Table 1. For example, in the first sequence shown in Table 1, the polymorphic site occupies the 16th base. The position can also be identified by reference to, for example, a chromosome, and distance from known markers within the chromosome. In other databases, information identifying a polymorphism contains sequences of 10-100 bases shown in Table 1 or the complements thereof, including a polymorphic site. Preferably, such information records at least 10, 15, 20, or 30 contiguous bases of sequences including a polymorphic site.

EXAMPLES The polymorphisms shown in Table 1 were identified by resequencing of target sequences from eight unrelated individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays. The strategy and principles for design and use of such arrays are generally described in WO 95/11995. The strategy provides arrays of probes for analysis of target sequences showing a high degree of sequence identity to the reference sequences of the fragments shown in Table 1, column 1. The reference sequences were sequence-tagged sites (STSs) developed in the course of the Human Genome Project ( see, e . g. , Science 270, 1945-1954 (1995); Nature 380, 152-154 (1996)). Most STS's ranged from 100 bp to 300 bp in size.

A typical probe array used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence. A first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences. Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two. For each probe in the first set, there are three corresponding probes from three additional probe sets . Thus, there are four probes corresponding to each nucleotide in the reference sequence. The probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets. In the present analysis, probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate .

Multiple target sequences from an individual were amplified from human genomic DNA using primers for the fragments indicated in the listed Web sites. The amplified target sequences were fluorescently labelled during or after PCR. The labelled target sequences were hybridized with a substrate bearing immobilized arrays of probes. The amount of label bound to probes was measured. Analysis of the pattern of label revealed the nature and position of differences between the target and reference sequence. For example, comparison of the intensities of four corresponding probes reveals the identity of a corresponding nucleotide in the target sequences aligned with the interrogation position of the probes . The corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity (see WO 95/11995) . The existence of a polymorphism is also manifested by differences in normalized hybridization intensities of probes flanking the polymorphism when the probes hybridized to corresponding targets from different individuals. For example, relative loss of hybridization intensity in a "footprint" of probes flanking a polymorphism signals a difference between the target and reference (i.e., a polymorphism) (see EP 717,113, incorporated by reference in its entirety for all purposes) . Additionally, hybridization intensities for corresponding targets from different individuals can be classified into groups or clusters suggested by the data, not defined a priori , such that isolates in a give cluster tend to be similar and isolates in different clusters tend to be dissimilar. See WO 97/29212 (incorporated by reference in its entirety for all purposes) . Hybridizations to samples from different individuals were performed separately. Table 1 summarizes the data obtained for target sequences in comparison with a reference sequence for the eight individuals tested. From the foregoing, it is apparent that the invention includes a number of general uses that can be expressed concisely as follows. The invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, such as cancer, inflammation, heart disease, diseases of the CNS, and susceptibility to infection by microorganisms. The invention further provides for the use of any of the nucleic acid segments in the manufacture of a medicament for the treatment or prophylaxis of such diseases. The invention further provides for the use of any of the DNA segments as a pharmaceutical .

All publications and patent applications cited above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incorporated by reference . Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims .

Claims

WHAT IS CLAIMED IS:

1 A nucleic acid segment of between 10 and 100 bases from a fragment shown in Table 1 including a polymorphic site, or the complement of the segment.

2. The nucleic acid segment of claim 1 that is DNA.

3. The nucleic acid segment of claim 1 that is RNA.

4. The segment of claim 1 that is less than 50 bases.

5. The segment of claim 1 that is less than 20 bases .

6. The segment of claim 1, wherein the fragment is WI-14263 and the polymorphic site is at position 49.

7. The segment of claim 1, wherein the polymorphic site is diallelic.

8. The segment of claim 1, wherein the polymorphic form occupying the polymorphic site is the reference base for the fragment listed in Table 1, column 3.

9. The segment of claim 1, wherein the polymorphic form occupying the polymorphic site is an alternative form for the fragment listed in Table 1, column 5.

10. An allele-specific oligonucleotide that hybridizes to a segment of a fragment shown in Table 1, column 8 or its complement .

11. The allele-specific oligonucleotide of claim 10 that is probe .

12. The allele-specific oligonucleotide of claim 10, wherein a central position of the probe aligns with the polymorphic site of the fragment .

13. The allele-specific oligonucleotide of claim 10 that is a primer.

14. The allele-specific oligonucleotide of claim 13, wherein the 3 ' end of the primer aligns with the polymorphic site of the fragment.

15. An isolated nucleic acid comprising a sequence of Table 1, column 8 or the complement thereof, wherein the polymorphic site within the sequence or complement is occupied by a base other than the reference base show in Table 1, column 3.

16. A method of analyzing a nucleic acid, comprising: obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymorphic sites shown in Table 1.

17. The method of claim 16, wherein the determining comprises determining a set of bases occupying a set of the polymorphic sites shown in Table 1.

18. The method of claim 16, wherein the nucleic acid is obtained from a plurality of individuals, and a base occupying one of the polymorphic positions is determined in each of the individuals, and the method further comprising testing each individual for the presence of a disease phenotype, and correlating the presence of the disease phenotype with the base.

19. A computer-readable storage medium for storing data for access by an application program being executed on a data processing system, comprising: a data structure stored in the computer-readable storage medium, the data structure including information resident in a database used by the application program and including: a plurality of records, each record of the plurality comprising information identifying a polymorphisms shown in Table 1.

20. The computer-readable storage medium of claim 19, wherein each record has a field identifying a base occupying a polymorphic site and a location of the polymorphic site.

21. The computer-readable storage medium of claim 19, wherein each record identifies a nucleic acid segment of between 10 and 100 bases from a fragment shown in Table 1 including a polymorphic site, or the complement of the segment.

22. The computer-readable storage medium of claim 19, comprising at least 10 records, each record comprising information identifying a different polymorphism shown in Table 1.

23. The computer- eadable storage medium of claim 19, comprising at least 100 records, each record comprising information identifying a different polymorphisms shown in Table 1.

24. A signal carrying data for access by an application program being executed on a data processing system, comprising: a data structure encoded in the signal, said data structure including information resident in a database used by the application program and including: a plurality of records, each record of the plurality comprising information identifying a polymorphism shown in Table 1.