Background

1.1. DNA - Deoxyribonucleic Acid

In humans, as in other higher organisms, a DNA molecule consists of two strands that wrap around each other

to resemble a twisted ladder whose sides, made of sugar and phosphate molecules, are connected by rungs of

nitrogen containing chemicals called bases. Four different bases are present in DNA: adenine (A), thymine (T),

cytosine (C), and guanine (G). The particular order of the bases arranged along the sugar- phosphate backbone

is called the DNA sequence; the sequence specifies the exact genetic instructions required to create a particular

organism with its own unique traits. The two DNA strands are held together by weak bonds between the bases

on each strand, forming base pairs (bp). Genome size is usually stated as the total number of base pairs; the

human genome contains roughly 3 billion bp. A gene is a segment of a DNA molecule (ranging from fewer

than 1 thousand bases to several million), located in a particular position on a specific chromosome, whose

base sequence contains the information necessary for protein synthesis.

1.2. RNA

RNA has the same structure as DNA. The primary differences between RNA and DNA are:

RNA has a hydroxyl group on the second carbon of the sugar and instead of using nucleotide thymine, RNA

uses another nucleotide called uracil (U). Since RNA has extra hydroxyl group on it's sugar strand, RNA is too

bulky to form a stable double helix therefore it exists as a single-stranded molecule. In addition to that, because

the RNA molecule is not restricted to a rigid double helix, it can form many different structures. There are

several different kinds of RNA made by the cell. They are mRNA, tRNA, rRNA and snRNA.
1.3. Proteins

Proteins are involved in almost all biological activities, structural or enzymatic. A protein is made by arranging

amino acids together in a specific sequence (the sequence of every protein is different). These amino acids are

held together by a special bond called a peptide bond. There are altogether 20 different amino acids.

1.4. The Central Dogma Of Molecular Biology

How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? This concept

is explained by the central dogma of molecular biology, according to which

 The DNA replicates its information in a process called replication that involves many enzymes.

 The DNA codes for the production of messenger RNA (mRNA) during transcription. In eukaryotic

cells, the mRNA is processed (essentially by splicing) and migrates from the nucleus to the cytoplasm.

 Messenger RNA carries coded information to ribosomes. The ribosomes "read" this information and

use it for protein synthesis. This process is called translation.

Diagrammatically,

CCTGAGCCAACTATTGATG

DNA

transcription

CCUGAGCCAACUAUUGAUG
mRNA

translation

Protein

PEPTIDE

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1.5. Protein structure

A striking characteristic of proteins is that they have very well defined 3-D structures. A stretched-out

polypeptide chain has no biological activity, and protein function arises from the conformation of the protein,

which is the 3-D arrangement or shape of the molecules in the protein. The native conformation of a protein

is determined by a number of factors, and the most important are the 4 levels of structure found in proteins.

Primary, secondary and tertiary refer to the molecules in a single polypeptide chain, and the fourth

(quaternary) refers to the interaction of several polypeptide chains to form a multi-chained protein. In this

paper, we limit our discussion to just the primary and secondary structure.

Primary Structure

The primary structure of a protein is determined by the number and order

of amino acids within a polypeptide chain. A polypeptide is a sequence of

two or more amino acids joined together by peptide bonds. Determination

of primary structure is an essential step in the characterization of a protein.

Secondary Structure

Protein secondary structure refers to regular, repeated patters of folding of the protein backbone. The two most

common folding patterns are the alpha helix and the beta sheet. Patterns result from regular hydrogen bond

patterns of backbone atoms.

In the alpha helix, the polypeptide folds by twisting into a right handed

screw so that all the amino acids can form hydrogen bonds with each other.

This high amount of hydrogen bonding stabilizes the structure so that it

forms a very strong rod-like structure.

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 The beta-pleated sheet is substantially different from the alpha-helix in that

it is a sheet rather than a rod and polypeptide chain is fully stretched rather

than tightly coiled as in helix. It is called a beta-pleated sheet because of

zig zag appearance when viewed from the side.

The tertiary structure of a protein is formed when the attractions of side chains and those of the secondary

structure combine and cause the amino acid chain to form a distinct and unique 3-dimensional structure. It is

this unique structure that gives a protein its specific function.

1.6. Multiple Sequence Alignment

Multiple alignment is the process of aligning two or more sequences with

each other in order to determine any evolutionary relationships. For

aligning two sequences the dynamic programming approach is the most

suitable. This approach can be generalized for multiple sequence alignment

also. But for a large number of sequences this approach becomes

impractical. There are heuristic methods available to speed up the dynamic

programming approach like the local multiple alignment using the Sum of Pairs scoring function. In our treatise,

we will show how HMMs can be effective in solving this problem.

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2. Hidden Markov Model (HMM) Architecture

2.1. Markov Chains

Let the three states of weather be Sunny, Cloudy and Rainy. We cannot expect these three weather states to

follow each other deterministically, but we might still hope to model the system that generates a weather pattern.

One way to do this is to assume that the state of the model depends only upon the previous states of the model.

This is called the Markov assumption and simplifies problems greatly. When considering the weather, the

Markov assumption presumes that today's weather can always be predicted solely given knowledge of the

weather of the past few days.

A Markov process is a process, which moves from state to

state depending (only) on the previous n states. The process

Rain is called an order n model where n is the number of states

affecting the choice of next state. The simplest Markov

process is a first order process, where the choice of state is

shows all possible first order transitions between the states

of the weather example.

The state transition matrix below shows possible transition probabilities for the weather example;

that is, if it was sunny yesterday, there is a probability of 0.5 that it will be sunny today, and 0.25 that it will be

cloudy or rainy.

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To initialize such a system, we need to state what the weather was (or probably was) on the day after creation;

we define this in a vector of initial probabilities, called the vector.

So, we know it was sunny on day 1.

We have now defined a first order Markov process consisting of :

 states : Three states - sunny, cloudy, rainy.

 vector : Defining the probability of the system being in each of the states at time 0.

 state transition matrix : The probability of the weather given the previous day's weather.

Any system that can be described in this manner is a Markov process.

2.2. Hidden Markov Models

In some cases the patterns that we wish to find are not described sufficiently by a Markov process. Returning

to the weather example, a hermit for instance may not have access to direct weather observations, but does

have a piece of seaweed. Folklore tells us that the state of the seaweed is probabilistically related to the state of

the weather - the weather and seaweed states are closely linked. In this case we have two sets of states

 observable states (the state of the seaweed) and

 hidden states (the state of the weather).

We wish to devise an algorithm for the hermit to forecast weather from the seaweed and the Markov

assumption without actually ever seeing the weather. The diagram below shows the hidden and observable

states in the weather example. It is assumed that the hidden states (the true weather) are modeled by a simple

first order Markov process, and so they are all connected to each other.

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 The connections between the hidden states and the

observable states represent the probability of

generating a particular observed state given that the

Markov process is in a particular hidden state. It

should thus be clear that all probabilities `entering'

an observable state will sum to 1, since in the above

case it would be the sum of Pr(Obs|Sun),

Pr(Obs|Cloud) and Pr(Obs|Rain).

In addition to the probabilities defining the Markov process, we therefore have another matrix, termed the

output matrix, which contains the probabilities of the observable states given a particular hidden state. For

the weather example the output matrix might be;

So, this is a model containing three sets of probabilities in addition to the two sets of states

 vector : contains the probability of the hidden model being in a particular hidden state at time t= 1.

 state transition matrix : holding the probability of a hidden state given the previous hidden state.

 output matrix : containing the probability of observing a particular observable state given that the

hidden model is in a particular hidden state.

Thus a hidden Markov model is a standard Markov process augmented by a set of observable states, and some

probabilistic relations between them and the hidden states.

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2.3. An example of a HMM for Protein Sequences

Transition

Prob.

Output Prob.

This is a possible hidden Markov model for the protein ACCY. The protein is represented as a sequence of

probabilities. The numbers in the boxes show the probability that an amino acid occurs in a particular state,

and the numbers next to the directed arcs show probabilities, which connect the states. The probability of

ACCY is shown as a highlighted path through the model. There are three kinds of states represented by three

different shapes. The squares are called match states, and the amino acids emitted from them form the

conserved primary structure of a protein. These amino acids are the same as those in the common ancestor or,

if not, are the result of substitutions. The diamond shapes are insert states and emit amino acids that result

from insertions. The circles are special, silent states known as delete states and model deletions. These type

of HMMs are called Protein Profile-HMMs and will be covered in more depth in the later sections.

Scoring a Sequence with an HMM

Any sequence can be represented by a path through the model. The probability of any sequence, given the

model, is computed by multiplying the emission and transition probabilities along the path. A path through

the model represented by ACCY is highlighted. For example, the probability of A being emitted in position 1

is 0.3, and the probability of C being emitted in position 2 is 0.6. The probability of ACCY along this path is

.4*.3*.46*.6*.97*.5*.015*.73*.01*1 = 1.76x10-6.

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2.4. Three Problems Of Hidden Markov Models

1) Scoring Problem

We want to find the probability of an observed sequence given an HMM. It can be seen that one method of

calculating the probability of the observed sequence would be to find each possible sequence of the hidden

states, and sum these probabilities. We use the Forward Algorithm for this.

I0
I1
M1
M3
M2

Consider the HMM shown above. In this figure several paths exist for the protein sequence ACCY.

The Forward algorithm employs a matrix, shown below. The columns of the matrix are indexed by the states

in the model, and the rows are indexed by the sequence. The elements of the matrix are initialized to zero and

then computed with these steps:

1. The probability that the amino acid A was generated by state I0 is computed and entered as the first element

of the matrix. This is .4*.3 = .12

2. The probabilities that C is emitted in state M1 (multiplied by the probability of the most likely transition to

state M1 from state I0) and in state I1 (multiplied by the most likely transition to state I1 from state I0) are

entered into the matrix element indexed by C and I1/M1.

3. The sum of the two probabilities, sum(I1, M1), is calculated.

4. A pointer is set from the winner back to state I0.

5. Steps 2-4 are repeated until the matrix is filled.

The probability of the sequence is found by summing the probabilities in the last column.

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Matrix for the Forward algorithm

2) Alignment Problem

We often wish to take a particular HMM, and determine from an observation sequence the most likely sequence

of underlying hidden states that might have generated it. This is the alignment problem and the Viterbi

Algorithm is used to solve this problem.

The Viterbi algorithm is similar to the forward algorithm. However in step 3, maximum rather than a sum is

calculated. The most likely path through the model can now be found by following the back-pointers.

Matrix for the Viterbi algorithm

Once the most probable path through the model is known, the probability of a sequence given the model can

be computed by multiplying all probabilities along the path.

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3) Training Problem

Another tricky problem is how to create an HMM in the first place, given a particular set of related training

sequences. It is necessary to estimate the amino acid emission distributions in each state and all state-to-state

transition probabilities from a set of related training sequences. This is done by using the Baum-Welch

Algorithm or the Forward Backward Algorithm.

The algorithm proceeds by making an initial guess of the parameters (which may well be entirely wrong) and

then refining it by assessing its worth, and attempting to reduce the errors it provokes when fitted to the given

data. In this sense, it is performing a form of gradient descent, looking for a minimum of an error measure.

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3. Applications of HMM’s

In this section, we will delve into greater depth at specific problems in the area of computational biology and

examine the role of HMM's.

3.1. Gene finding and prediction

We introduce here the gene-prediction HMMs that can be used to predict the structure of the gene. Our

objective is to find the coding and non-coding regions of an unlabeled string of DNA nucleotides.

The motivation behind this is to

 assist in the annotation of genomic data produced by genome sequencing methods

 gain insight into the mechanisms involved in transcription, splicing and other processes

As shown in the diagram above, a string of DNA nucleotides containing a gene will have separate regions

 Introns – non-coding regions within a gene

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  Exons – coding regions

These regions are separated by functional sites

 Start and stop codons

 Splice sites – acceptors and donors

In the process of transcription, only the exons are left to form the protein sequence as depicted below.

Many problems in biological sequence analysis have a grammatical structure . HMMs are very useful in modeling

grammar. The input to such a HMM is the genomic DNA sequence and the output, in the simplest case is a

parse tree of exons and introns on the DNA sequence.

Shown below is a simple model for unspliced genes that recognizes the start codon, stop codon (only one of

the three possible stop codons are shown) and the coding/non-coding regions. This model has been trained

with a test set of gene data.

Having such a model, how can we predict genes in a sequence of anonymous DNA ? We simply use the Viterbi

algorithm to find the most probable path through the model

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3.2. Protein- Profile HMMs

As we have seen earlier, protein structural similarities make it possible to create a statistical model of a protein

family which is called a profile. The idea is, given a single amino acid target sequence of unknown structure,

we want to infer the structure of the resulting protein. The profile HMM is built by analyzing the distribution

of amino-acids in a training set of related proteins. This HMM in a natural way can model positional dependant

gap penalties.

The basic topology of a profile HMM is shown above. Each position, or module, in the model has three states.

Matching Deletion

Insertion
A state shown as a rectangular box is a match state that models the distribution of letters in the corresponding

column of an alignment. A state shown by a diamond-shaped box models insertions of random letters between

two alignment positions, and a state shown by a circle models a deletion, corresponding to a gap in an

alignment. States of neighboring positions are connected, as shown by lines. For each of these lines there is an

associated `transition probability', which is the probability of going from one state to the other.

The match state represents a consensus amino acid for this position in the protein family. The delete state is a

non-emitting state, and represents skipping this consensus position in the multiple alignment. Finally, the insert

state models the insertion of any number of residues after this consensus position.

A repository of protein profile HMMs can be found in the PFAM Database (http://pfam.wustl.edu).

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Building profiles from a family of proteins(or DNA) a profile HMM can be made for searching a database for

other members of the family. As we have seen before in the section on HMM problems, profile HMM’s can

also be used for the following

Scoring a sequence

We are calculating the probability of a sequence given a profile by simply multiplying emmision and transition

probabilities along the path.

Classifying sequences in a database

Given a HMM for a protein family and some unknown sequences, we are trying to find a path through the

model where the new sequence fits in or we are tying to ‘align’ the sequence to the model. Alignment to the

model is an assignment of states to each residue in the sequence. There are many such alignments and the

Vitterbi’s algorithm is used to give the probability of the sequence for that alignment.

Creating Multiple sequence alignment

HMMs can be used to automatically create a multiple alignment from a group of unaligned sequences. By taking

a close look at the alignment, we can see the history of evolution. One great advantage of HMMs is that they

can be estimated from sequences, without having to align the sequences first. The sequences used to estimate

or train the model are called the training sequences, and any reserved sequences used to evaluate the model are

called the test sequences. The model estimation is done with the forward-backward algorithm, also known as

the Baum-Welch algorithm. It is an iterative algorithm that maximizes the likelihood of the training sequences.

3.3. Prediction of protein secondary structure using HMM’s

Prediction of secondary structures is need for the prediction of protein function. As an alternative method to

direct X-ray analysis, a HMM is used to

 Analyze the amino-acid sequences of proteins

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  Learn secondary structures such as helix, sheet and turn

 Predict the secondary structures of sequences

The method is to train the four HMMs of secondary structure – helix, sheet, turn and other – by training

sequences. The Baum-Welch method is used to train the HMMs. So, the HMM of helix is able to produce

helix-like sequences with high probabilities. Now, these HMMs can be used to predict the secondary structure

of the test sequence. The forward-backward algorithm is used to compute the probabilities of these HMMs

outputting the test sequence. The sequence has the secondary structure whose HMM showed the highest

probability to output the sequence.

4. HMM implementation

These are the two publicly available HMM implementation software.

HMMER - http://hmmer.wustl.edu/

SAM system - http://www.cse.ucsc.edu/research/compbio/sam.html

5. Advantages of HMMs

 HMM’s can accommodate variable-length sequence.

Because most biological data has variable-length properties, machine learning techniques which require a fixed-

length input, such as neural networks or support vector machines, are less successful in biological sequence

analysis

 Allows position dependant gap penalties.

HMM’s treat insertions and deletions is a statistical manner that is dependant on position.

6. Limitations of HMMs

 Linear Model

So, they are unable to capture higher order correlations among amino-acids.

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 Markov Chain assumption of independent events

Probabilities of states are supposed to be independent which is not true of biology

Eg, P(y) must be independent of P(x), and vice versa

P(x … P(y

 Standard Machine Learning Problems

In the training problem, we need to watch out for local maxima and so model may not converge to a truly

optimal parameter set for a given training set. Secondly, since the model is only as good as your training set,

this may lead to over-fitting.

7. Open areas for research in HMMs in biology

 Integration of structural information into profile HMMs.

Despite the almost obvious application of using structural information on a member protein family when one

exists to better the parameterization of the HMM, this has been extremely hard to achieve in practice.

 Model architecture

The architectures of HMMs have largely been chosen to be the simplest architectures that can fit the observed

data. Is this the best architecture to use? Can one use protein structure knowledge to make better architecture

decisions, or, in limited regions, to learn the architecture directly from the data? Will these implied architectures

have implications for our structural understanding?

 Biological mechanism

In gene prediction, the HMM’s may be getting close to replicating the same sort of accuracy as the biological

machine (the HMM’s have the additional task of finding the gene in the genomic DNA context, which is not

handled by the biological machine that processes the RNA). What constraints does our statistical model place

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on the biological mechanism— in particular, can we consider a biological mechanism that could use the same

information as the HMM?

8. References

1. L. R. Rabiner and B. H. Juang, An Introduction to Hidden Markov Models, IEEE ASSP Magazine, January 1986,

pp. 1-16.

2. K. Asai, S. Hayamizu and H. Handa, Prediction of protein secondary structures by hidden Markon models, Computer

Application in the Biosciences (CABIOS), Vol 9, No 2, 1993, pp. 141-146.

3. Krogh, A., M. Brown, I.S. Mian, K. Sjolander, and D. Haussler. Hidden Markov Models in Computational

Biology: Applications to Protein Modeling, J. Mol. Biol., Vol. 235, pp.1501-1531, 1994.

4. S. Eddy. Profile hidden Markov models. Bioinformatics, 14:755--763, 1998.

5. L. R. Rabiner, A Tutorial on Hidden Markov Models and Selected Applications in Speech Recognition , Proceedings

of the IEEE, 77 , no. 2, 257--285, February 1989.

6. Baldi, P., Chauvin, Y., Hunkapiller, T. and McClure, M.A. (1993) Hidden Markov Models in Molecular Biology:

New Algorithms and Applications, In Advances in Neural Information Processing Systems 5, Eds. S.J. Hanson,

J.D. Cowan and C. Lee Giles, (Morgan Kaufmann) pp 747-754

7. Baldi, P., Chauvin, Y., Hunkapiller, T., and McClure, M.A. (1994) Hidden Markov Models of Biological Primary

Sequence Information, Proceedings of the National Academy of Science, USA 91: 1059-1063.

8. David Kulp, David Haussler, Martin G. Reese, and Frank H. Eeckman, A generalized hidden markov model for

the recognition of human genes in DNA, Procedings of the Fourth International Conference on Intelligent

Systems for Molecular Biology (Menlo Park, CA), AAAI Press, 1996.

9. R. Hughey and A. Krogh. Hidden Markov models for sequence analysis: extension and analysis of the basic

method. Computer Applications in the Biosciences, 12:95-107.1996

http://www.cse.ucsc.edu/research/compbio/html_format_papers/hughkrogh96/cabios.html

10. Henderson,J., Salzberg,S. and Fasman,K. Finding genes in human DNA with a hidden Markov model. Journal of

Computational Biology 1997, 4, 127--141.

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11. An Introduction to Hidden Markov Models for Biological Sequences by A. Krogh. In S. L. Salzberg et al., eds.,

Computational Methods in Molecular Biology, 45-63. Elsevier, 1998.

12. E Birney. Hidden Markov models in biological sequence analysis. Deep computing for the life sciences.

Volume 45, Numbers ¾ 2001. http://www.research.ibm.com/journal/rd/453/birney.html

13. HMMer - http://hmmer.wustl.edu/

14. SAM system - http://www.cse.ucsc.edu/research/compbio/sam.html

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