Overview of Ibis Forensic Analysis Software

Transcription

1 Thomas Hall, Ph.D. Overview of Ibis Forensic Analysis Software

2 Outline General software interface Features Data processing Data analysis mtdna SNPs Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 2

3 Forensic Analysis Software Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 3

4 Software Features Utilizes IbisTrack database software for sample tracking Integrated system MitoDNA analysis module is an independent module Updates to MitoDNA Simply place updated module with most recent IbisTrack; integration is dynamic Provides functions for: Analysis / QC of assay data and registration of profiles Adding / removing databases, populations, sequence profiles, and base composition profiles Automated conversion of sequence to mtdna base count profiles Population based searching and reporting for mtdna Plate based analysis reporting for mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 4

5 Data Processing Processor automation monitor MassCollapse (raw processing) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 5

6 Forensic Data Processing Monitor Mass spectrometer controller queues plate for processing Data are processed with MassCollapse and output is imported into database TCP/IP Channel DB Data Processor MassCollapse Processing monitor works through database TCP/IP Channel Viewer monitors queue and triggers processing in request order Data Analysis Trigger Monitor Processing Viewer Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 6

7 MassCollapse Deconvolution Raw spectrum Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 7

8 MassCollapse Deconvolution Raw spectrum Deconvolved spectrum Signal intensity ,000 32,000 34,000 36,000 38,000 40,000 42,000 44,000 Mass (Da) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 8

9 MassCollapse Deconvolution Raw spectrum Signal intensity ,000 32,000 34,000 36,000 38,000 40,000 42,000 44,000 Mass (Da) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 9

10 MassCollapse Deconvolution Raw spectrum Signal intensity List of masses and intensities Collapsed data can also be interrogated for fit of base compositions given a model of the Ibis mass tag 0 30,000 32,000 34,000 36,000 38,000 40,000 42,000 44, Da Da Da Da Da Da Mass (Da) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 10

11 Forensic Analysis Software IbisTrack with forensic analysis MassCollapse (raw processing) Processor automation monitor Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 11

12 Basic Application Interface Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 12

13 Basic Application Interface Main IbisTrack program menu Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 13

14 Basic Application Interface mtdna Analysis menu Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 14

15 Basic Application Interface STR / Y STR Analysis Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 15

16 Basic Application Interface Autosomal SNP Analysis Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 16

17 Basic Application Interface mtdna Click Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 17

18 Basic Application Interface mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 18

19 Basic Application Interface mtdna Set time stamp filters Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 19

20 Basic Application Interface mtdna Select an assay plate Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 20

21 Basic Application Interface mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 21

22 Basic Application Interface mtdna Select sample Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 22

23 Basic Application Interface mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 23

24 Basic Application Interface mtdna Click Analyze Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 24

25 Basic Application Interface mtdna Plate selection Sample selection Plate timestamp selection Interface navigation Sample / well navigation Coverage map Product assignment info Spectral schematic views Base count profile Mass assignment detail Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 25

26 Basic Application Interface mtdna Click Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 26

27 Basic Utilities Interface mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 27

28 Basic Application Interface mtdna Item addition Item deletion Sample arrangement Database maintenance Mass data import Example: Single addition of a sequence profile Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 28

29 Basic Application Interface mtdna Item addition Item deletion Sample arrangement Database maintenance Mass data import Sequence imports can be done by: 1. Manual profile entry (or copy/paste) 2. Import of raw sequence (software aligns and creates profile automatically) 3. Import LISA exported profiles (e.g. AFDIL system) 4. Import CMF formatted profiles (can be done in batch) Conversion to base composition profiles is automatic for all Example: Single addition of a sequence profile Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 29

30 Sample Analysis mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 30

31 Sample Analysis mtdna Set time stamp filters Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 31

32 Sample Analysis mtdna Select an assay plate Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 32

33 Sample Analysis mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 33

34 Sample Analysis mtdna Select sample Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 34

35 Sample Analysis mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 35

36 Sample Analysis mtdna Click Analyze Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 36

37 Single Sample Analysis mtdna Single assignment bottom strand Mass measurement error (ppm) Single assignment top strand ppm = Parts per million: 1 ppm = expected mass / 1,000,000 ppm error = ((expected observed)/expected) * 1,000,000 Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 37

38 Single Sample Analysis mtdna Single assignment top strand Single assignment bottom strand Product strand corresponds to a mass peak Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 38

39 Single Sample Analysis mtdna Single assignment top strand Profile record Primer pair CRS coords base count Single assignment bottom strand Product strand corresponds to a mass peak Pair of masses corresponds to a doublestranded PCR product Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 39

40 Single Sample Analysis mtdna Three products in a well correspond to three roughly evenly spaced products Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 40

41 Use of a Mass Tag High mass precision and mass tag combine to provide unambiguous base compositions in routine operation A48 G18 C31 T Da Da Some double SNPs cause small mass differences Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 41

42 Use of a Mass Tag Without mass tag: Product strands differ by 1 Da for two products that differ by a G A and C T SNP at the same time A47 G19 C32 T Da Da A48 G18 C31 T Da Da Some double SNPs cause small mass differences Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 42

43 Use of a Mass Tag With mass tag: With 13 C dgtp, the mass separation increases to ~10 Da for each strand; this is about 300 ppm (we generally see errors ~10 ppm) A mass tag increases mass separation for these SNPs A47 G19 C32 T Da Da Corresponding = A48 G18 C31 T40 untagged peaks Da Da Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 43

44 Real Data with Mass Tag Primer pair 2906 product from two representative samples (data for primer pair 2906 are overlayed) Well A05: P A47 G19 C32 T39 Well A01: P Da Da A48 G18 C31 T Da Da Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 44

45 What Does Mass Tagging Mean? For any base count For all combinations of base differences plus or minus 10 A, G, C, T Ax Gx Cx 10 Tx Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 45

46 What Does Mass Tagging Mean? For all combinations of base differences plus or minus 10 A, G, C, T And increasing measurement error For increasing product length Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 46

47 What Does Mass Tagging Mean? Potential assignment ambiguities increase dramatically with measurement error and product size Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 47

48 What Does Mass Tagging Mean? Ambiguous assignments not a problem in our normal working range of mass measurement Working range for TOF (<20 ppm) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 48

49 What Does Mass Tagging Mean? Most assignments would be fine with natural nucleotides. Some assignments fall outside of normal range. Working range for TOF (<20 ppm) Which 6% of measurements are a problem? Percentages are based upon 123,415 mass assignments Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 49

50 Mass Tag Changes Rules With 13 C dgtp, the mass separation increases to about 10 Da for each strand; this is about 300 ppm (we generally see errors about 10 ppm) A47 G19 C32 T Da Da A48 G18 C31 T Da Da Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 50

51 What Does Mass Tagging Mean? Using 13 C dgtp eliminates ambiguity with a wide margin around our working zone 70 Maximum error tolerance without ambiguity is here Our largest product is here Working range for TOF (<20 ppm) 80 ppm measurement error maximum in this assay Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 51

52 What Does Mass Tagging Mean? Using 13 C dgtp eliminates ambiguity with a wide margin around our working zone 70 Maximum error tolerance without ambiguity is here Our largest product is here Working range for TOF (<20 ppm) 80 ppm measurement error maximum in this assay Threshold at 70 ppm to be conservative Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 52

53 Spectral Viewer Used to examine data quality, signal, noise, or to take a picture of the spectrum Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 53

54 Spectral Viewer Deconvolved Zoom view can give an idea of data quality surrounding a given peak Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 54

55 Base Composition Browser Integrated base composition browser can be opened from the spectral viewer Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 55

56 Base Composition Browser Primer pairs in current well are integrated to this view Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 56

57 Base Composition Browser Major product is automatically shown for primer choice Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 57

58 Base Composition Browser All known products for selected primer pair are in this list Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 58

59 Base Composition Browser If base composition is changed, dynamic overlays placed where product would be if it were detected Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 59

60 Base Composition Browser Adenylation products, sodium adducts, and potassium adducts can also be looked for here Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 60

61 Search Database mtdna Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 61

62 Search Database mtdna - Max number of differences to show in interface C length heteroplasmy treatment Target databases Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 62

63 Search Database mtdna Target populations choice Target database Target populations Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 63

64 Search Database mtdna Min differences between query and target Ave min differences over entire population Search output Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 64

65 Comparing mtdna Profiles AF : : no data 2925: : no data 2899: : no data 2898: : A15 G7 C17 T : : A15 G8 C14 T : : A27 G7 C27 T : : A17 G1 C19 T8 2893: : A24 G5 C28 T : : A17 G4 C22 T8 N 2891: : A23 G5 C18 T15 N 2890: : no data 2889: : no data 2902: 5..97: no data 2903: : no data 2904: : A10 G10 C20 T : : A23 G9 C21 T : : A32 G15 C15 T : : A30 G6 C30 T : : A25 G6 C35 T : : no data 2910: : no data 2916: : no data 2912: : no data 2913: : no data X USA.FBI : : no data 2925: : no data 2899: : A7 G10 C5 T : : A15 G7 C16 T : : A15 G8 C14 T : : A26 G7 C29 T : : A16 G1 C21 T7 2893: : A23 G5 C29 T : : A18 G4 C22 T8 2891: : A24 G5 C19 T : : A8 G5 C17 T : : A12 G9 C20 T : : A5 G15 C12 T : : A12 G24 C21 T : : A9 G11 C18 T : : A22 G10 C19 T : : A32 G15 C15 T : : A30 G6 C31 T : : A25 G6 C36 T : : A27 G6 C37 T : : A21 G3 C14 T9 2916: : A11 G7 C12 T : : A20 G1 C34 T : : A27 G6 C46 T5 diff 2 X 2 X 1 X 3 X 3 Total minimum differences 6 1 Accounting for overlaps Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 65

66 Profile Registration mtdna Analysis of a full plate exposes profile registration interface Samples that pass auto processing are checked by default Controls are not checked by default Samples that failed will be disabled If positive or negative control fails, registration for entire plate will be disabled; disabled state can be overridden Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 66

67 Report Generation mtdna Click to generate a report Log general plate comments here Log samplespecific comments here Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 67

68 mtdna Plate Analysis Report Page 1 First page of report summarizes samples and controls on a plate Quantity (or dilution) used Status Ave abundance (for controls) Ave error (for positive) Control status Samples that have registered profiles User comments Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 68

69 mtdna Plate Analysis Report Page 2 Second page of report summarizes analysis parameters used Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 69

70 mtdna Plate Analysis Report Summary Page Each sample gets a summary page Coverage map Average abundance Average error Status Comparison to other samples on the plate Log of manual assignments and filtered assignments (if any) User comments Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 70

71 mtdna Plate Analysis Report Assignment Report At the end of the report is a comprehensive raw mass to product assignment report in table format for the entire plate that serves as a raw data log Every report generated is automatically archived into the database Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 71

72 SNP Sample Analysis Data processing is automated and utilizes the same code as mtdna data processing SNP analysis interface is similar to mtdna interface Allele names displayed (as SNP identities) instead of base compositions Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 72

73 SNP Sample Analysis Highlighted allele assignment details Homozygous Heterozygous Allele assignment Spectral thumbnails Sample profile Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 73

74 SNP Sample Analysis Plate Profile Summary Profiles can be registered to the database here Profiles can be copied to the clipboard here Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 74

75 SNP Sample Analysis Profile Query Select Profiles Select Assay Select Database Select Population Copy and/or save profile table Select Samples Query Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 75

76 Summary Data processing after MS is automated Processed data persist in database with registered sample and plate information Sample analysis is automated (triggered manually) Interface provides dynamic graphical functions to evaluate, QC, and adjust (if necessary) profile assignments Profiles can be registered a plate at a time Query profiles from database Automated comparison of profiles to database entries (mtdna) Search database (mtdna) Add sequences and convert to base compositions in the database (mtdna) Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 76

77 Questions? Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 77

78 Contact Information Thomas Hall Ibis Biosciences division of Abbott Molecular Note: All images and charts courtesy of Tom Hall, Ph.D. Forensic SNP Analysis Overview of Ibis Forensic Analysis Software 78