sequence alignment. cs262 lecture 3, win06, batzoglou sequence alignment...

Sequence Alignment

CS262 Lecture 3, Win06, Batzoglou

Sequence Alignment

-AGGCTATCACCTGACCTCCAGGCCGA--TGCCC---TAG-CTATCAC--GACCGC--GGTCGATTTGCCCGAC

DefinitionGiven two strings x = x1x2...xM, y = y1y2…yN,

an alignment is an assignment of gaps to positions0,…, N in x, and 0,…, N in y, so as to line up each

letter in one sequence with either a letter, or a gapin the other sequence

AGGCTATCACCTGACCTCCAGGCCGATGCCCTAGCTATCACGACCGCGGTCGATTTGCCCGAC


The Needleman-Wunsch Algorithm

1. Initialization.F(0, 0) = F(0, j) = F(i, 0) = 0

2. Main Iteration. a. For each i = 1……M

For each j = 1……N F(i-1,j-1) + s(xi,

yj) [case 1] F(i, j) = max F(i-1, j) – d [case 2] F(i, j-1) – d [case 3]

DIAG, if [case 1] Ptr(i,j) = LEFT, if [case 2]

UP, if [case 3]

3. Termination. F(M, N) is the optimal score, andfrom Ptr(M, N) can trace back optimal alignment


The Smith-Waterman algorithm

Idea: Ignore badly aligning regions

Modifications to Needleman-Wunsch:

Initialization: F(0, j) = F(i, 0) = 0

0

Iteration: F(i, j) = max F(i – 1, j) – d

F(i, j – 1) – d

F(i – 1, j – 1) + s(xi, yj)


Scoring the gaps more accurately

Simple, linear gap model:Gap of length nincurs penalty nd

However, gaps usually occur in bunches

Convex gap penalty function:(n):for all n, (n + 1) - (n) (n) - (n – 1)

Algorithm: O(N3) time, O(N2) space

(n)

(n)


Compromise: affine gaps

(n) = d + (n – 1)e | | gap gap open extend

To compute optimal alignment,

At position i, j, need to “remember” best score if gap is open best score if gap is not open

F(i, j): score of alignment x1…xi to y1…yj

ifif xi aligns to yj

G(i, j): score ifif xi aligns to a gap after yj

H(i, j): score ifif yj aligns to a gap after xi

V(i, j) = best score of alignment x1…xi to y1…yj

de

(n)


Needleman-Wunsch with affine gaps

Why do we need matrices F, G, H?

• xi aligns to yj

x1……xi-1 xi xi+1

y1……yj-1 yj -

2. xi aligns to a gap

x1……xi-1 xi xi+1

y1……yj …- -

Add -d

Add -e

G(i+1, j) = V(i, j) – d

G(i+1, j) = G(i, j) – e

Because, perhaps

G(i, j) < V(i, j)

(it is best to align xi to yj if we were aligningonly x1…xi to y1…yj and not the rest of x, y),

but on the contrary

G(i, j) – e > V(i, j) – d

(i.e., had we “fixed” our decision that xi alignsto yj, we could regret it at the next step whenaligning x1…xi+1 to y1…yj)


Needleman-Wunsch with affine gaps

Initialization: V(i, 0) = d + (i – 1)eV(0, j) = d + (j – 1)e

Iteration:V(i, j) = max{ F(i, j), G(i, j), H(i, j) }

F(i, j) = V(i – 1, j – 1) + s(xi, yj)

V(i – 1, j) – d G(i, j) = max

G(i – 1, j) – e

V(i, j – 1) – d H(i, j) = max

H(i, j – 1) – e

Termination: V(i, j) has the best alignment

Time?Space?


To generalize a little…

… think of how you would compute optimal alignment with this gap function

….in time O(MN)

(n)


Bounded Dynamic Programming

Assume we know that x and y are very similar

Assumption: # gaps(x, y) < k(N)

xi Then, | implies | i – j | < k(N)

yj

We can align x and y more efficiently:

Time, Space: O(N k(N)) << O(N2)


Bounded Dynamic Programming

Initialization:

F(i,0), F(0,j) undefined for i, j > k

Iteration:

For i = 1…M

For j = max(1, i – k)…min(N, i+k)

F(i – 1, j – 1)+ s(xi, yj)

F(i, j) = max F(i, j – 1) – d, if j > i – k(N)

F(i – 1, j) – d, if j < i + k(N)

Termination: same

Easy to extend to the affine gap case

x1 ………………………… xM

y1 …

……

……

……

……

…

yN

k(N)


Linear-Space Alignment


Subsequences and Substrings

Definition A string x’ is a substring of a string x,if x = ux’v for some prefix string u and suffix string v

(similarly, x’ = xi…xj, for some 1 i j |x|)

A string x’ is a subsequence of a string xif x’ can be obtained from x by deleting 0 or more letters

(x’ = xi1…xik, for some 1 i1 … ik |x|)

Note: a substring is always a subsequence

Example: x = abracadabray = cadabr; substringz = brcdbr; subseqence, not substring


Hirschberg’s algortihm

Given a set of strings x, y,…, a common subsequence is a string u that is a subsequence of all strings x, y, …

• Longest common subsequence Given strings x = x1 x2 … xM, y = y1 y2 … yN, Find longest common subsequence u = u1 … uk

• Algorithm:F(i – 1, j)

• F(i, j) = max F(i, j – 1)F(i – 1, j – 1) + [1, if xi = yj; 0 otherwise]

• Ptr(i, j) = (same as in N-W)

• Termination: trace back from Ptr(M, N), and prepend a letter to u whenever • Ptr(i, j) = DIAG and F(i – 1, j – 1) < F(i, j)

• Hirschberg’s algorithm solves this in linear space


F(i,j)

Introduction: Compute optimal score

It is easy to compute F(M, N) in linear space

Allocate ( column[1] )

Allocate ( column[2] )

For i = 1….M

If i > 1, then:

Free( column[i – 2] )

Allocate( column[ i ] )

For j = 1…N

F(i, j) = …


Linear-space alignment

To compute both the optimal score and the optimal alignment:

Divide & Conquer approach:

Notation:

xr, yr: reverse of x, y

E.g. x = accgg;

xr = ggcca

Fr(i, j): optimal score of aligning xr1…xr

i & yr1…yr

j

same as aligning xM-i+1…xM & yN-j+1…yN



Lemma: (assume M is even)

F(M, N) = maxk=0…N( F(M/2, k) + Fr(M/2, N-k) )

x

y

M/2

k*

F(M/2, k) Fr(M/2, N-k)



• Now, using 2 columns of space, we can compute

for k = 1…M, F(M/2, k), Fr(M/2, N-k)

PLUS the backpointers



• Now, we can find k* maximizing F(M/2, k) + Fr(M/2, N-k)

• Also, we can trace the path exiting column M/2 from k*

k*

k*+1

0 1 …… M/2 M/2+1 …… M M+1



• Iterate this procedure to the left and right!

N-k*

M/2M/2

k*



Hirschberg’s Linear-space algorithm:

MEMALIGN(l, l’, r, r’): (aligns xl…xl’ with yr…yr’)

1. Let h = (l’-l)/22. Find (in Time O((l’ – l) (r’-r)), Space O(r’-r))

the optimal path, Lh, entering column h-1, exiting column hLet k1 = pos’n at column h – 2 where Lh enters

k2 = pos’n at column h + 1 where Lh exits

3. MEMALIGN(l, h-2, r, k1)

4. Output Lh

5. MEMALIGN(h+1, l’, k2, r’)

Top level call: MEMALIGN(1, M, 1, N)



Time, Space analysis of Hirschberg’s algorithm: To compute optimal path at middle column,

For box of size M N,Space: 2NTime: cMN, for some constant c

Then, left, right calls cost c( M/2 k* + M/2 (N-k*) ) = cMN/2

All recursive calls cost Total Time: cMN + cMN/2 + cMN/4 + ….. = 2cMN = O(MN)

Total Space: O(N) for computation,O(N+M) to store the optimal alignment


Heuristic Local Alignerers

1. The basic indexing & extension technique

2. Indexing: techniques to improve sensitivityPairs of Words, Patterns

3. Systems for local alignment


State of biological databases

http://www.cbs.dtu.dk/databases/DOGS/

~10x per3 years


State of biological databases

• Number of genes in these genomes:

Mammals: ~24,000 Insects: ~14,000 Worms: ~17,000 Fungi: ~6,000-10,000

Small organisms: 100s-1,000s

• Each known or predicted gene has one or more associated protein sequences

• >1,000,000 known / predicted protein sequences


Some useful applications of alignments

• Given a newly discovered gene, Does it occur in other species? How fast does it evolve?

• Assume we try Smith-Waterman:

The entire genomic database

Our new gene

104

1010 - 1012


Some useful applications of alignments

• Given a newly sequenced organism,• Which subregions align with other organisms?

Potential genes Other biological characteristics

• Assume we try Smith-Waterman:

The entire genomic database

Our newly sequenced mammal

3109

1010 - 1012


Indexing-based local alignment

(BLAST- Basic Local Alignment Search Tool)

Main idea:

1. Construct a dictionary of all the words in the query

2. Initiate a local alignment for each word match between query and DB

Running Time: O(MN)

However, orders of magnitude faster than Smith-Waterman

query

DB


Indexing-based local alignment

Dictionary:

All words of length k (~10)

Alignment initiated between words of alignment score T

(typically T = k)

Alignment:

Ungapped extensions until score

below statistical threshold

Output:

All local alignments with score

> statistical threshold

……

……

query

DB

query

scan


Indexing-based local alignment—Extensions

A C G A A G T A A G G T C C A G T

C

C

C

T

T

C C

T

G

G

A T

T

G

C

G

A

Example:

k = 4

The matching word GGTC initiates an alignment

Extension to the left and right with no gaps until alignment falls < C below best so far

Output:

GTAAGGTCC

GTTAGGTCC




C

T

G

A

T

C C

T

G

G

A

T

T

G C

G

A

Gapped extensions

• Extensions with gaps in a band around anchor

Output:

GTAAGGTCCAGTGTTAGGTC-AGT




C

T

G

A

T

C C

T

G

G

A

T

T

G C

G

A

Gapped extensions until threshold

• Extensions with gaps until score < C below best score so far

Output:

GTAAGGTCCAGTGTTAGGTC-AGT


Sensitivity-Speed Tradeoff

long words

(k = 15)

short words

(k = 7)

Sensitivity Speed

Kent WJ, Genome Research 2002

Sens.

Speed

X%


Sensitivity-Speed Tradeoff

Methods to improve sensitivity/speed

1. Using pairs of words

2. Using inexact words

3. Patterns—non consecutive positions

……ATAACGGACGACTGATTACACTGATTCTTAC……

……GGCACGGACCAGTGACTACTCTGATTCCCAG……

……ATAACGGACGACTGATTACACTGATTCTTAC……

……GGCGCCGACGAGTGATTACACAGATTGCCAG……

TTTGATTACACAGAT T G TT CAC G


Measured improvement

Kent WJ, Genome Research 2002


Non-consecutive words—Patterns

Patterns increase the likelihood of at least one match within a long conserved region

3 common

5 common

7 common

Consecutive Positions Non-Consecutive Positions

6 common

On a 100-long 70% conserved region: Consecutive Non-consecutive

Expected # hits: 1.07 0.97Prob[at least one hit]: 0.30 0.47


Advantage of Patterns

11 positions

11 positions

10 positions


Multiple patterns

• K patterns Takes K times longer to scan Patterns can complement one another

• Computational problem: Given: a model (prob distribution) for homology between two regions Find: best set of K patterns that maximizes Prob(at least one match)

TTTGATTACACAGAT T G TT CAC G T G T C CAG TTGATT A G

Buhler et al. RECOMB 2003Sun & Buhler RECOMB 2004

How long does it take to search the query?


Variants of BLAST

• NCBI BLAST: search the universe http://www.ncbi.nlm.nih.gov/BLAST/• MEGABLAST: http://genopole.toulouse.inra.fr/blast/megablast.html

Optimized to align very similar sequences• Works best when k = 4i 16• Linear gap penalty

• WU-BLAST: (Wash U BLAST) http://blast.wustl.edu/ Very good optimizations Good set of features & command line arguments

• BLAT http://genome.ucsc.edu/cgi-bin/hgBlat Faster, less sensitive than BLAST Good for aligning huge numbers of queries

• CHAOS http://www.cs.berkeley.edu/~brudno/chaos Uses inexact k-mers, sensitive

• PatternHunter http://www.bioinformaticssolutions.com/products/ph/index.php Uses patterns instead of k-mers

• BlastZ http://www.psc.edu/general/software/packages/blastz/ Uses patterns, good for finding genes

• Typhon http://typhon.stanford.edu Uses multiple alignments to improve sensitivity/speed tradeoff


Example

Query: gattacaccccgattacaccccgattaca (29 letters) [2 mins]

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS, or phase 0, 1 or 2 HTGS sequences) 1,726,556 sequences; 8,074,398,388 total letters

>gi|28570323|gb|AC108906.9| Oryza sativa chromosome 3 BAC OSJNBa0087C10 genomic sequence, complete sequence Length = 144487 Score = 34.2 bits (17), Expect = 4.5 Identities = 20/21 (95%) Strand = Plus / Plus

Query: 4 tacaccccgattacaccccga 24 ||||||| |||||||||||||

Sbjct: 125138 tacacccagattacaccccga 125158

Score = 34.2 bits (17),

Expect = 4.5 Identities = 20/21 (95%) Strand = Plus / Plus



>gi|28173089|gb|AC104321.7| Oryza sativa chromosome 3 BAC OSJNBa0052F07 genomic sequence, complete sequence Length = 139823 Score = 34.2 bits (17), Expect = 4.5 Identities = 20/21 (95%) Strand = Plus / Plus




Example

Query: Human atoh enhancer, 179 letters [1.5 min]

Result: 57 blast hits1. gi|7677270|gb|AF218259.1|AF218259 Homo sapiens ATOH1 enhanc... 355 1e-95 2. gi|22779500|gb|AC091158.11| Mus musculus Strain C57BL6/J ch... 264 4e-68 3. gi|7677269|gb|AF218258.1|AF218258 Mus musculus Atoh1 enhanc... 256 9e-66 4. gi|28875397|gb|AF467292.1| Gallus gallus CATH1 (CATH1) gene... 78 5e-12 5. gi|27550980|emb|AL807792.6| Zebrafish DNA sequence from clo... 54 7e-05 6. gi|22002129|gb|AC092389.4| Oryza sativa chromosome 10 BAC O... 44 0.068 7. gi|22094122|ref|NM_013676.1| Mus musculus suppressor of Ty ... 42 0.27 8. gi|13938031|gb|BC007132.1| Mus musculus, Similar to suppres... 42 0.27

gi|7677269|gb|AF218258.1|AF218258 Mus musculus Atoh1 enhancer sequence Length = 1517 Score = 256 bits (129), Expect = 9e-66 Identities = 167/177 (94%),

Gaps = 2/177 (1%) Strand = Plus / Plus Query: 3 tgacaatagagggtctggcagaggctcctggccgcggtgcggagcgtctggagcggagca 62 ||||||||||||| ||||||||||||||||||| |||||||||||||||||||||||||| Sbjct: 1144 tgacaatagaggggctggcagaggctcctggccccggtgcggagcgtctggagcggagca 1203

Query: 63 cgcgctgtcagctggtgagcgcactctcctttcaggcagctccccggggagctgtgcggc 122 |||||||||||||||||||||||||| ||||||||| |||||||||||||||| ||||| Sbjct: 1204 cgcgctgtcagctggtgagcgcactc-gctttcaggccgctccccggggagctgagcggc 1262

Query: 123 cacatttaacaccatcatcacccctccccggcctcctcaacctcggcctcctcctcg 179 ||||||||||||| || ||| |||||||||||||||||||| |||||||||||||||

Sbjct: 1263 cacatttaacaccgtcgtca-ccctccccggcctcctcaacatcggcctcctcctcg 1318 http://www.ncbi.nlm.nih.gov/BLAST/


The Four-Russian Algorithmbrief overview

A (not so useful) speedup of Dynamic Programming

[Arlazarov, Dinic, Kronrod, Faradzev 1970]


Main Observation

Within a rectangle of the DP matrix,values of D depend onlyon the values of A, B, C,

and substrings xl...l’, yr…r’

Definition: A t-block is a t t square of

the DP matrix

Idea: Divide matrix in t-blocks,Precompute t-blocks

Speedup: O(t)

A B

C

D

xl xl’

yr

yr’

t


The Four-Russian Algorithm

Main structure of the algorithm:

1. Divide NN DP matrix into KK log2N-blocks that overlap by 1 column & 1 row

2. For i = 1……K

3. For j = 1……K

4. Compute Di,j as a function of Ai,j, Bi,j, Ci,j, x[li…l’i], y[rj…r’j]

Time: O(N2 / log2N)

times the cost of step 4t t t


The Four-Russian Algorithm

t t t

sequence alignment. cs262 lecture 3, win06, batzoglou sequence alignment...

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