Overview of the Ibis SNP Assay

Transcription

1 Thomas Hall, Ph.D. Overview of the Ibis SNP ssay

2 Objective PR/ESI MS based assay for human autosomal SNP analysis Exclude non contributors to a DN sample random profile match should have very low probability Minimize population bias Multiple markers with about 50% heterozygosity Low Fst (distribution same in all populations) Low detectable genetic linkage (low linkage disequilibrium) Use product rule for probability estimates Use global q or no q correction for population substructure 40 independent markers Forensic SNP nalysis Overview of the Ibis SNP ssay 2

3 Ideal Bi allelic Markers One from father One from mother Each autosomal chromosome comes as a pair B B B Bi allelic markers have one of two states ny marker for any individual can be both or both B or one of each Forensic SNP nalysis Overview of the Ibis SNP ssay 3

4 Ideal Bi allelic Markers There s a 50% chance of getting either allele on the father s chromosome B nd a 50% chance of getting either allele on the mother s chromosome Each marker is like a coin toss Forensic SNP nalysis Overview of the Ibis SNP ssay 4

5 Ideal Bi allelic Markers There s a 50% chance of getting either allele on the father s chromosome nd a 50% chance of getting either allele on the mother s chromosome Each marker is like a coin toss That means 50% x 50% = 25% chance of getting two heads ( allele) Forensic SNP nalysis Overview of the Ibis SNP ssay 5

6 Ideal Bi allelic Markers There s a 50% chance of getting either allele on the father s chromosome B B nd a 50% chance of getting either allele on the mother s chromosome Each marker is like a coin toss nd 50% x 50% = 25% chance of getting two tails (B allele) Forensic SNP nalysis Overview of the Ibis SNP ssay 6

7 Ideal Bi allelic Markers B Each marker is like a coin toss nd 50% x 50% = 25% chance of getting + B Forensic SNP nalysis Overview of the Ibis SNP ssay 7

8 Ideal Bi allelic Markers B B Each marker is like a coin toss nd 50% x 50% = 25% chance of getting + B Plus 50% x 50% = 25% chance of getting B + = 50% chance of being heterozygous Forensic SNP nalysis Overview of the Ibis SNP ssay 8

9 Ideal Bi allelic Markers For any two individuals, the random odds they will match at any one locus is 25% x 25% = 6.25 % + 25% x 25% = 6.25 % + 25% x 25% = 6.25 % B B + 25% x 25% = 6.25 % B B + 25% x 25% = 6.25 % + 25% x 25% = 6.25 % = 37.5 % or probability of Forensic SNP nalysis Overview of the Ibis SNP ssay 9

10 Ideal Bi allelic Markers Random match probability for one marker Random match probability for another marker X = Random match probability for two marker profile Forensic SNP nalysis Overview of the Ibis SNP ssay 10

11 Ideal Bi allelic Markers Random match probability for four marker profile = x x x = = lose to 99% with four perfectly distributed bi allelic markers Forensic SNP nalysis Overview of the Ibis SNP ssay 11

12 Ideal Bi allelic Markers With 40 unlinked and perfectly distributed SNPs, the random match probability would ideally be , or 9.15 x Forensic SNP nalysis Overview of the Ibis SNP ssay 12

13 Kidd 40 SNP Panel Large collection of SNP positions with data for three major population groups Subpanel identified with low Fst and high heterozygosity Evaluated subpanel over seven populations 73 SNPs with Fst < 0.02 over seven populations 40 final SNPs with Fst < 0.06 and heterozygosity > 0.4 across 40 populations around the world Reference: Pakstis,., Speed, W., Kidd, J., Kidd, K. andidate SNPs for a Universal Individual Identification Panel. Human enetics 121 (3 4) (May 2007): Forensic SNP nalysis Overview of the Ibis SNP ssay 13

14 The Kidd pproach 90,483 SNPs from BI catalog Frequency data for European, frican and hinese/japanese Forensic SNP nalysis Overview of the Ibis SNP ssay 14

15 The Kidd pproach 90,483 SNPs from BI catalog 436 SNPs with low F st and high heterozygosity Informatically selected 436 SNPs 73 SNPs accepted for 7 populations Tested in TaqMan SNP assays across 7 populations 73 SNPs Tested across 40 populations 40 SNPs Final reduced panel Forensic SNP nalysis Overview of the Ibis SNP ssay 15

16 Kidd Population overage 2070 total individuals 40 total populations Several major global regions lobal F st < 0.06 all populations verage heterozygosity > 0.4 Median LD = (ave = 0.029) Forensic SNP nalysis Overview of the Ibis SNP ssay 16

17 Resolving Power of Kidd SNP Panel Perfect 40 SNP panel: random match with probability of 9.15 x Kidd 40 panel has ave of about 1 x match probability Within populations, ave match probability ranged from to Number of occurrences Number of matching loci between two individuals 1,568 full profiles 40 populations ll pairwise profile to profile comparisons 2,457,056 pairwise comparisons Most people will differ at loci Forensic SNP nalysis Overview of the Ibis SNP ssay 17

18 Why Use Mass Spectrometry? Unified platform for major DN forensics applications Mitochondrial DN profiling STR analysis utosomal SNP analysis SNPs, STRs and / or mtdn could be analyzed automatically on one instrument in the same run High degree of accuracy Potential rare variant will be resolved rather than missed (could a T position ever present an or a?) Forensic SNP nalysis Overview of the Ibis SNP ssay 18

19 Initial Development: Eight 5 plex Reactions dd 5 ml template to each well of a plate and thermocycle RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS Each sample is distributed across one column of an assay plate Forensic SNP nalysis Overview of the Ibis SNP ssay 19

20 Reaction 1 Initial Multiplexing Results RS Well 1 (01) RS N ng RS RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \1\acqus RS RS Well 3 (03) RS S ng RS RS , RS \\ibisfs.ibisbio.com\mtdnadata\data\p \3\acqus Reaction RS RS Well 13 (B01) \\ibisfs.ibisbio.com\mtdnadata\data\p \13\acqus RS RS , T RS T RS Well 15 (B03) RS , RS T \\ibisfs.ibisbio.com\mtdnadata\data\p \15\acqus RS , T RS T Forensic SNP nalysis Overview of the Ibis SNP ssay 20

21 Initial Multiplexing Results Reaction RS T N ng Well 25 (01) RS RS RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \25\acqus RS S ng Well 27 (03) RS RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \27\acqus RS Reaction RS Well 37 (D01) RS RS RS , T RS \\ibisfs.ibisbio.com\mtdnadata\data\p \37\acqus RS Well 39 (D03) RS RS RS T RS \\ibisfs.ibisbio.com\mtdnadata\data\p \39\acqus Forensic SNP nalysis Overview of the Ibis SNP ssay 21

22 Initial Multiplexing Results Reaction RS , N ng Well 49 (E01) \\ibisfs.ibisbio.com\mtdnadata\data\p \49\acqus RS RS RS RS RS , S ng Well 51 (E03) \\ibisfs.ibisbio.com\mtdnadata\data\p \51\acqus RS RS RS RS Reaction RS RS T RS Well 61 (F01) RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \61\acqus RS RS RS RS , RS , Well 63 (F03) \\ibisfs.ibisbio.com\mtdnadata\data\p \63\acqus RS Forensic SNP nalysis Overview of the Ibis SNP ssay 22

23 Initial Multiplexing Results Reaction RS Well 73 (01) \\ibisfs.ibisbio.com\mtdnadata\data\p \73\acqus RS315791, N ng RS RS RS RS Well 75 (03) RS S ng \\ibisfs.ibisbio.com\mtdnadata\data\p \75\acqus RS RS RS Reaction RS RS RS Well 85 (H01) \\ibisfs.ibisbio.com\mtdnadata\data\p \85\acqus RS RS RS RS T RS Well 87 (H03) \\ibisfs.ibisbio.com\mtdnadata\data\p \87\acqus RS RS Forensic SNP nalysis Overview of the Ibis SNP ssay 23

24 Initial Intra locus Strand Balance Percentage of total assigned signal Forensic SNP nalysis Overview of the Ibis SNP ssay 24

25 Reaction 1 djusted Multiplexing Results RS Well 1 (01) RS N31774 RS RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \1\acqus RS RS Well 3 (03) RS S35495 RS RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \3\acqus RS Reaction RS RS Well 13 (B01) RS RS , T RS T \\ibisfs.ibisbio.com\mtdnadata\data\p \13\acqus RS Well 15 (B03) RS , RS T RS , T \\ibisfs.ibisbio.com\mtdnadata\data\p \15\acqus RS T Forensic SNP nalysis Overview of the Ibis SNP ssay 25

26 Reaction 3 djusted Multiplexing Results RS T Well 25 (01) \\ibisfs.ibisbio.com\mtdnadata\data\p \25\acqus RS RS RS RS Well 27 (03) RS \\ibisfs.ibisbio.com\mtdnadata\data\p \27\acqus RS RS N31774S35495 RS RS Reaction RS RS RS Well 37 (D01) RS , T RS \\ibisfs.ibisbio.com\mtdnadata\data\p \37\acqus RS Well 39 (D03) RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \39\acqus RS T RS Forensic SNP nalysis Overview of the Ibis SNP ssay 26

27 Reaction 5 djusted Multiplexing Results RS , N31774 Well 49 (E01) RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \49\acqus RS RS RS , S35495 Well 51 (E03) RS RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \51\acqus RS Reaction RS RS T RS Well 61 (F01) RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \61\acqus RS RS Well 63 (F03) \\ibisfs.ibisbio.com\mtdnadata\data\p \63\acqus RS RS , RS , RS Forensic SNP nalysis Overview of the Ibis SNP ssay 27

28 Reaction 7 djusted Multiplexing Results RS Well 73 (01) \\ibisfs.ibisbio.com\mtdnadata\data\p \73\acqus RS315791, N31774 RS RS RS RS Well 75 (03) RS S35495 RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \75\acqus RS Reaction RS RS RS RS Well 85 (H01) \\ibisfs.ibisbio.com\mtdnadata\data\p \85\acqus RS RS RS T RS Well 87 (H03) \\ibisfs.ibisbio.com\mtdnadata\data\p \87\acqus RS RS Forensic SNP nalysis Overview of the Ibis SNP ssay 28

29 Improved Intra locus Strand Balance RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS RS B01 B02 B03 B D01 D02 D03 D04 E01 E02 E03 E04 F01 F02 F03 F H01 H02 H03 H04 Percentage of total assigned signal N31774 S35495 N31774 S35495 N31774 S35495 N31774 S35495 N31774 S35495 N31774 S35495 N31774 S35495 N31774 S35495 Rxn 1 Rxn 2 Rxn 3 Rxn 4 Rxn 5 Rxn 6 Rxn 7 Rxn 8 Well number Forensic SNP nalysis Overview of the Ibis SNP ssay 29

30 Sensitivity 5 plexes Full profile obtained at 63 pg / reaction Reaction quality degraded at 125 pg and below Forensic SNP nalysis Overview of the Ibis SNP ssay 30

31 Inter Locus 5 plex Balance in Dilutions 125 pg 125 pg 125 = pg decreasing 125 pg template 125 concentration pg 125 pg 125 pg 125 pg Balance was most consistent down to about 125 pg/reaction, then became highly variable. Drop outs occurred after 63 pg/reaction Forensic SNP nalysis Overview of the Ibis SNP ssay 31

32 10 plex Reactions Reaction RS ng/reaction N31774 DN RS RS RS RS , RS RS RS , T Well 3 (03) :\MitoProject\Human_SNPS\Reports\data\P \3\acqus RS T RS Reaction 2 RS T RS RS RS RS RS RS Well 15 (B03) :\MitoProject\Human_SNPS\Reports\data\P \15\acqus RS RS , T RS Forensic SNP nalysis Overview of the Ibis SNP ssay 32

33 10 plex Reactions Reaction RS , RS ng/reaction N31774 DN RS RS RS RS RS RS Well 27 (03) :\MitoProject\Human_SNPS\Reports\data\P \27\acqus RS , RS Reaction RS RS315791, RS RS RS RS RS RS RS Well 39 (D03) :\MitoProject\Human_SNPS\Reports\data\P \39\acqus RS Forensic SNP nalysis Overview of the Ibis SNP ssay 33

34 Inter Locus Balance in 10 plex Reactions Reaction 1 Reaction 2 Reaction 3 Reaction 4 SD = % SD = % SD = % SD = % Forensic SNP nalysis Overview of the Ibis SNP ssay 34

35 Sensitivity of 10 plex Reactions Full profile at 62.5 pg / reaction Forensic SNP nalysis Overview of the Ibis SNP ssay 35

36 Species Specificity Human blood derived DN sample was tested in duplicate in the presence of 10 fold excess of exogenous DN 1 ng of human DN per reaction 10 ng exogenous DN Dog (male merican Eskimo buccal swab) at (male long hair, buccal swab) andida albicans (yeast) spergillus oryzae (environmental filamentous fungus) Escherichia coli (gram negative bacterium) Staphylococcus aureus (gram positive bacterium) ll tests with exogenous DN gave a full profile Forensic SNP nalysis Overview of the Ibis SNP ssay 36

37 Reaction 1 Species Specificity RS With dog DN Well 1 (01) RS RS RS , RS \\ibisfs.ibisbio.com\mtdnadata\data\p \1\acqus With spergillus oryzae DN RS RS Well 3 (03) RS RS , RS \\ibisfs.ibisbio.com\mtdnadata\data\p \3\acqus Reaction RS Well 13 (B01) RS , RS T RS , T \\ibisfs.ibisbio.com\mtdnadata\data\p \13\acqus RS T RS Well 15 (B03) RS T \\ibisfs.ibisbio.com\mtdnadata\data\p \15\acqus RS , RS , T RS T Forensic SNP nalysis Overview of the Ibis SNP ssay 37

38 Species Specificity With dog DN With spergillus oryzae DN Reaction RS Well 25 (01) \\ibisfs.ibisbio.com\mtdnadata\data\p \25\acqus RS RS RS RS RS Well 27 (03) \\ibisfs.ibisbio.com\mtdnadata\data\p \27\acqus RS RS RS RS Reaction RS Well 37 (D01) RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \37\acqus RS T RS RS RS RS Well 39 (D03) \\ibisfs.ibisbio.com\mtdnadata\data\p \39\acqus RS T RS Forensic SNP nalysis Overview of the Ibis SNP ssay 38

39 Species Specificity With dog DN With spergillus oryzae DN Reaction RS , Well 49 (E01) \\ibisfs.ibisbio.com\mtdnadata\data\p \49\acqus RS RS RS RS RS , Well 51 (E03) \\ibisfs.ibisbio.com\mtdnadata\data\p \51\acqus RS RS RS RS Reaction RS RS RS , Well 61 (F01) RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \61\acqus RS RS RS RS , Well 63 (F03) RS , \\ibisfs.ibisbio.com\mtdnadata\data\p \63\acqus RS Forensic SNP nalysis Overview of the Ibis SNP ssay 39

40 Reaction 7 Species Specificity RS With dog DN Well 73 (01) \\ibisfs.ibisbio.com\mtdnadata\data\p \73\acqus RS RS RS RS With spergillus oryzae DN RS Well 75 (03) \\ibisfs.ibisbio.com\mtdnadata\data\p \75\acqus RS RS RS RS Reaction RS Well 85 (H01) RS T RS RS RS \\ibisfs.ibisbio.com\mtdnadata\data\p \85\acqus RS RS T RS Well 87 (H03) \\ibisfs.ibisbio.com\mtdnadata\data\p \87\acqus RS RS Forensic SNP nalysis Overview of the Ibis SNP ssay 40

41 Species Specificity 5 plex 10 fold excess of bacterial DN 1 (01) (02) (03) (04) (05) (06) (07) (08) (09) (10) (11) (12) Mass (B01) (Da) Mass (B02) (Da) Mass (B03) (Da) Mass (B04) (Da) Mass (B05) (Da) Mass (B06) (Da) Mass (B07) (Da) Mass (B08) (Da) Mass (B09) (Da) Mass (B10) (Da) Mass (B11) (Da) Mass (B12) (Da) Mass (01) (Da) Mass (02) (Da) Mass (03) (Da) Mass (04) (Da) Mass (05) (Da) Mass (06) (Da) Mass (07) (Da) Mass (08) (Da) Mass (09) (Da) Mass (10) (Da) Mass (11) (Da) Mass (12) (Da) Mass (D01) (Da) Mass (D02) (Da) Mass (D03) (Da) Mass (D04) (Da) Mass (D05) (Da) Mass (D06) (Da) Mass (D07) (Da) Mass (D08) (Da) Mass (D09) (Da) Mass (D10) (Da) Mass (D11) (Da) Mass (D12) (Da) Mass (E01) (Da) Mass (E02) (Da) Mass (E03) (Da) Mass (E04) (Da) Mass (E05) (Da) Mass (E06) (Da) Mass (E07) (Da) Mass (E08) (Da) Mass (E09) (Da) Mass (E10) (Da) Mass (E11) (Da) Mass (E12) (Da) Mass (F01) (Da) Mass (F02) (Da) Mass (F03) (Da) Mass (F04) (Da) Mass (F05) (Da) Mass (F06) (Da) Mass (F07) (Da) Mass (F08) (Da) Mass (F09) (Da) Mass (F10) (Da) Mass (F11) (Da) Mass (F12) (Da) Mass (01) (Da) Mass (02) (Da) Mass (03) (Da) Mass (04) (Da) Mass (05) (Da) Mass (06) (Da) Mass (07) (Da) Mass (08) (Da) Mass (09) (Da) Mass (10) (Da) Mass (11) (Da) Mass (12) (Da) Mass (H01) (Da) Mass (H02) (Da) Mass (H03) (Da) Mass (H04) (Da) Mass (H05) (Da) Mass (H06) (Da) Mass (H07) (Da) Mass (H08) (Da) Mass (H09) (Da) Mass (H10) (Da) Mass (H11) (Da) Mass (H12) (Da) Forensic SNP nalysis Overview of the Ibis SNP ssay 41

42 Species Specificity 10 plex 10 fold excess of fungal DN Forensic SNP nalysis Overview of the Ibis SNP ssay 42

43 Reaction 1 Species Specificity 10 plex ng human DN + 10 ng andida albicans (yeast) DN RS Well 5 (05) RS RS RS \\ibisna2.ibisbio.com\mtdnadata\data\p \5\acqus RS , RS RS RS , T RS T RS Reaction RS T RS RS Well 17 (B05) RS RS \\ibisna2.ibisbio.com\mtdnadata\data\p \17\acqus RS RS RS RS , T RS Forensic SNP nalysis Overview of the Ibis SNP ssay 43

44 Reaction 3 Species Specificity 10 plex RS , RS ng human DN + 10 ng andida albicans (yeast) DN Well 29 (05) RS RS RS RS T RS RS \\ibisna2.ibisbio.com\mtdnadata\data\p \29\acqus RS , RS Reaction RS RS RS315791, RS Well 41 (D05) \\ibisna2.ibisbio.com\mtdnadata\data\p \41\acqus RS RS RS RS RS RS Forensic SNP nalysis Overview of the Ibis SNP ssay 44

45 Species Specificity Full profiles were obtained for all replicates in the presence of 10 fold excess of exogenous DN from six different sources Forensic SNP nalysis Overview of the Ibis SNP ssay 45

46 Profile comparisons 86 samples run with Ibis Kidd-40 SNP panel 50 samples from UNTHS 26 blood samples 10 buccal swab samples ross comparisons of all pairs of samples: Samples differ at an average of loci 3655 pair-wise cross-comparisons Forensic SNP nalysis Overview of the Ibis SNP ssay 46

47 Resolving Power of Kidd SNP Panel Perfect 40 SNP panel: random match with probability of 9.15 x Kidd 40 panel has ave of about 1 x match probability Within populations, ave match probability ranged from to Number of occurrences Number of matching loci between two individuals 1,568 full profiles 40 populations ll pairwise profile to profile comparisons 2,457,056 pairwise comparisons Most people will differ at loci Forensic SNP nalysis Overview of the Ibis SNP ssay 47

48 ross omparisons of Ibis Profiles Number of occurrences ll pair wise comparisons between 86 individuals Distribution of comparisons for small set of 86 unrelated samples is virtually identical to 1568 samples taken from 40 global population groups Number of matching loci between 2 individuals Forensic SNP nalysis Overview of the Ibis SNP ssay 48

49 ross omparisons of Ibis Profiles ll pair wise comparisons between 1568 individuals 1000 Number of occurrences Kidd population samples Number of occurrences Ibis samples 0 0 Number of matching loci between 2 individuals Forensic SNP nalysis Overview of the Ibis SNP ssay 49

50 Reducing Spectral omplexity Multiple PR products congest an ESI mass spectrum Multiplexing capacity is limited both by PR and mass spectrometry Multiplexing capacity may be able to be increased by removing one PR product strand Removing one DN strand from each product would cut spectral complexity in half It may be possible to effectively double multiplexing capacity Forensic SNP nalysis Overview of the Ibis SNP ssay 50

51 Reducing Spectral omplexity By using one biotin tagged forward primer in each primer pair biotin nd an excess of nontagged reverse primer Template Forensic SNP nalysis Overview of the Ibis SNP ssay 51

52 Reducing Spectral omplexity PR products can be generated with an over abundance of non tagged reverse strands biotin Forensic SNP nalysis Overview of the Ibis SNP ssay 52

53 Reducing Spectral omplexity fter running PR Streptavidin coated magnetic beads are added Forensic SNP nalysis Overview of the Ibis SNP ssay 53

54 Reducing Spectral omplexity Biotin tags bind streptavidin beads after a short incubation Forensic SNP nalysis Overview of the Ibis SNP ssay 54

55 Reducing Spectral omplexity Magnetic beads are pulled to bottom of wells with a magnet Single DN strands are left to be analyzed by MS Forensic SNP nalysis Overview of the Ibis SNP ssay 55

56 Single-stranded Multiplex Products, Rxn 1 ontrol reaction RS RS RS RS , RS Biotin-tagged PR Single-strand depleted RS Biotin tagging one primer strand RS RS RS , RS Forensic SNP nalysis Overview of the Ibis SNP ssay 56

57 Single stranded Multiplex Profiles Double stranded products produced Single stranded products produced Forensic SNP nalysis Overview of the Ibis SNP ssay 57

58 Summary Basic 5 plex and 10 plex, 40 Kidd SNP assays defined and moving into validation Limit of sensitivity down to pg / reaction Mammal, fungal or bacterial DN does not appear to interfere with the assay Small panel of samples showed average locus differences comparable to Kidd results Integrated software developed and in process Forensic SNP nalysis Overview of the Ibis SNP ssay 58

59 Funded by FBI contract #J FBI Questions? Forensic SNP nalysis Overview of the Ibis SNP ssay 59

60 ontact Information Thomas Hall Ibis Biosciences division of bbott Molecular Note: ll images and charts courtesy of Tom Hall, Ph.D. Forensic SNP nalysis Overview of the Ibis SNP ssay 60