1. Mutivariate statistical Analysis methods Ahmed Rebaï Centre of Biotechnology of Sfax ahmed.rebai@cbs.rnrt.tn

2. Basic statistical concepts and tools

3. Statistics  Statistics are concerned with the ‘optimal’ methods of analyzing data generated from some chance mechanism (random phenomena).  ‘Optimal’ means appropriate choice of what is to be computed from the data to carry out statistical analysis

4. Random variables  A random variable is a numerical quantity that in some experiment, that involve some degree of randomness, takes one value from some set of possible values  The probability distribution is a set of values that this random variable takes together with their associated probability

5. The Normal distribution  Proposed by Gauss (1777-1855) : the distribution of errors in astronomical observations (error function)  Arises in many biological processes,  Limiting distribution of all random variables for a large number of observations.  Whenever you have a natural phenomemon which is the result of many contributiong factor each having a small contribution you have a Normal

6. The Quincunx Bell-shaped distribution

7. Distribution function  The distribution function is defined F(x)=Pr(X<x)  F is called the cumulative distribution function (cdf) and f the probability distrbution function (pdf) of X   and  ² are respectively the mean and the variance of the distribution

9. Moments of a distribution  The k th moment is defined as  The first moment is the mean  The k th moment about the mean  is  The second moment about the mean is called the variance  ²

10. Kurtosis: a useful moments’ function  Kurtosis  4 =  4 - 3  ² 2   4  0 for a normal distribution so it is a measure of Normality

11. Observations  Observations x i are realizations of a random variable X  The pdf of X can be visualized by a histogram: a graphics showing the frequency of observations in classes

12. Estimating moments  The Mean of X is estimated from a set of n observations (x 1, x 2,..x n ) as  The variance is estimated by Var(X) =

13. The fundamental of statistics  Drawing conclusions about a population on the basis on a set of measurments or observations on a sample from that population  Descriptive: get some conclusions based on some summary measures and graphics (Data Driven)  Inferential: test hypotheses we have in mind befor collecting the data (Hypothesis driven).

14. What about having many variables?  Let X=(X 1, X 2,..X p ) be a set of p variables  What is the marginal distribution of each of the variables X i and what is their joint distribution  If f(X 1, X 2,..X p ) is the joint pdf then the marginal pdf is

15. Independance  Variables are said to be independent if f(X 1, X 2,..X p )= f(X 1 ). f(X 2 )…. f(X p )

16. Covariance and correlation  Covariance is the joint first moment of two variables, that is Cov(X,Y)=E(X-  X )(Y-  Y )=E(XY)-E(X)E(Y)  Correlation: a standardized covariance   is a number between -1 and +1

17. For example: a bivariate Normal  Two variables X and Y have a bivariate Normal if   is the correlation between X and Y

19. Uncorrelatedness and independence  If  =0 (Cov(X,Y)=0) we say that the variables are uncorrelated  Two uncorrelated variables are independent if and only if their joint distribution is bivariate Normal  Two independent variables are necessarily uncorrelated

20. Bivariate Normal  If  =0 then  So f(x,y)=f(x).f(y)  the two variables are thus independent

21. Many variables  We can calculate the Covariance or correlation matrix of (X 1, X 2,..X p )  C=Var(X)=  A square ( p x p ) and symmetric matrix

22. A Short Excursion into Matrix Algebra

24. What is a matrix?

25. Operations on matrices Transpose

26. Properties

27. Some important properties

28. Other particular operations

29. Eigenvalues and Eigenvectors

30. Singular value decomposition

31. Multivariate Data

32.  Data for which each observation consists of values for more than one variables;  For example: each observation is a measure of the expression level of a gene i in a tissue j  Usually displayed as a data matrix

33. Biological profile data

35. The data matrix n observations (rows) for p variables (columns) an nxp matrix

36. Contingency tables  When observations on two categorial variables are cross-classified.  Entries in each cell are the number of individuals with the correponding combination of variable values Eyes colourHair colour FairRedMediumDark Blue32638241110 Medium34384909412 Dark9848403681 Light688116584188

37. Mutivariate data analysis

38. Exploratory Data Analysis  Data analysis that emphasizes the use of informal graphical procedures not based on prior assumptions about the structure of the data or on formal models for the data  Data= smooth + rough where the smooth is the underlying regularity or pattern in the data. The objective of EDA is to separate the smooth from the rough with minimal use of formal mathematics or statistics methods

39. Reduce dimensionality without loosing much information

40. Overview on the techiques  Factor analysis  Principal components analysis  Correspondance analysis  Discriminant analysis  Cluster analysis

41. Factor analysis A procedure that postulates that the correlations between a set of p observed variables arise from the relationship of these variables to a small number k of underlying, unobservable, latent variables, usually known as common factors where k<p

42. Principal components analysis  A procedure that transforms a set of variables into new ones that are uncorrelated and account for a decreasing proportions of the variance in the data  The new variables, named principal components (PC), are linear combinations of the original variables

43. PCA  If the few first PCs account for a large percentage of the variance (say >70%) then we can display the data in a graphics that depicts quite well the original observations

44. Example

46. Correspondance Analysis  A method for displaying relationships between categorial variables in a scatter plot  The new factors are combinations of rows and columns  A small number of these derived coordinate values (usually two) are then used to allow the table to be displayed graphically

47. Example: analysis of codon usage and gene expression in E. coli (McInerny, 1997) A gene can be represented by a 59- dimensional vector (universal code) A genome consists of hundreds (thousands) of these genes Variation in the variables (RSCU values) might be governed by only a small number of factors For each gene and each codon i calculate RCSU=# observed codon /#expected codon

48. Codon usage in bacterial genomes

49. Evidence that all synonymous codons were not used with equal frequency: Fiers et al., 1975 A-protein gene of bacteriophage MS2, Nature 256, 273-278 UUU Phe 6 UCU Ser 5 UAU Tyr 4 UGU Cys 0 UUC Phe 10 UCC Ser 6 UAC Tyr 12 UGC Cys 3 UUA Leu 8 UCA Ser 8 UAA Ter * UGA Ter * UUG Leu 6 UCG Ser 10 UAG Ter * UGG Trp 12 CUU Leu 6 CCU Pro 5 CAU His 2 CGU Arg 7 CUC Leu 9 CCC Pro 5 CAC His 3 CGC Arg 6 CUA Leu 5 CCA Pro 4 CAA Gln 9 CGA Arg 6 CUG Leu 2 CCG Pro 3 CAG Gln 9 CGG Arg 3 AUU Ile 1 ACU Thr 11 AAU Asn 2 AGU Ser 4 AUC Ile 8 ACC Thr 5 AAC Asn 15 AGC Ser 3 AUA Ile 7 ACA Thr 5 AAA Lys 5 AGA Arg 3 AUG MeU 7 ACG Thr 6 AAG Lys 9 AGG Arg 4 GUU Val 8 GCU Ala 6 GAU Asp 8 GGU Gly 15 GUC Val 7 GCC Ala 12 GAC Asp 5 GGC Gly 6 GUA Val 7 GCA Ala 7 GAA Glu 5 GGA Gly 2 GUG Val 9 GCG Ala 10 GAG Glu 12 GGG Gly 5

50. Multivariate reduction  Attempts to reduce a high-dimensional space to a lower-dimensional one. In other words, it tries to simplify the data set. Many of the variables might co-vary, therefore there might only be one, or a small few sources of variation in the dataset A gene can be represented by a 59-dimensional vector (universal code) A genome consists of hundreds (thousands) of these genes Variation in the variables (RSCU values) might be governed by only a small number of factors

51. Plot of the two most important axes Highly expressed genes Lowly-expressed genes Recently acquired genes

52. Discriminant analysis  Techniques that aim to assess whether or a not a set of variables distinguish or discriminate between two or more groups of individuals  Linear discriminant analysis (LDA): uses linear functions (called canonical discriminant functions) of variable giving maximal separation between groups (assumes tha covariance matrices within the groups are the same)  if not use Quadratic Discriminant analysis (QDA)

53. Example: Internal Exon prediction  Data: A set of exons and non-exons Variables : a set of features  donor/acceptor site recognizers  octonucleotide preferences for coding region  octonucleotide preferences for intron interiors  on either side

54. LDA or QDA

55. Cluster analysis  A set of methods (hierarchical clustering, K-means clustering,..) for constructing sensible and informative classification of an initially unclassified set of data  Can be used to cluster individuals or variables

56. Example: Microarray data

57. Other Methods  Independant component analysis (ICA): similar to PCA but components are defined as independent and not only uncorrelated; moreover they are not orthogonal and uniquely defined  Multidimensional Scaling (MDS): a clustering technique that construct a low-dimentional geometrical representation of a distance matrix (also Principal coordinates analysis)

58. Useful books: Data analysis

60. Useful book: R langage