martijn vermaat department of human genetics center for ......alignment methods sequence alignment...

Alignment methods

Martijn Vermaat

Department of Human Genetics

Center for Human and Clinical Genetics

Alignment methods

Sequence alignment

Assembly vs alignment

Alignment methods

Common issues

Platform specifics

Software

Metagenomics course 1/28 Thursday, 7 February 2013

Sequence alignment

Identifying regions of similarity in sequences


Sequence alignment

Identifying regions of similarity in sequences

In NGS

• Recovering original nucleotide sequence

• . . . from many short fragments

• . . . using a known reference


Sequence alignment

Pairwise alignment


Sequence alignment

Multiple sequence alignment


Sequence alignment

Global vs local alignment


Sequence alignment

Structural alignment



Alignment methods

Sequence alignment


Alignment methods

Common issues

Platform specifics

Software



Assembly



Assembly

Alignment



Assembly

• Memory hungry

• Needs high coverage



Assembly

• Memory hungry

• Needs high coverage

Alignment

• Easy to do in parallel

• Restricted by reference sequence

• highly polymorphic regions• large insertions


Alignment methods

Alignment methods

Sequence alignment


Alignment methods

Common issues

Platform specifics

Software


Alignment methods

Smith-Waterman

• Generalization of Needleman-Wunsch• Guaranteed optimal alignment

− A C A C A C T A

− 0 0 0 0 0 0 0 0 0

A 0 2 1 2 1 2 1 0 2

G 0 1 1 1 1 1 1 0 1

C 0 0 3 2 3 2 3 2 1

A 0 2 2 5 4 5 4 3 4

C 0 1 4 4 7 6 7 6 5

A 0 2 3 6 6 9 8 7 8

C 0 1 4 5 8 8 11 10 9

A 0 2 3 6 7 10 10 10 12

gap penalty = −1

match = +2

mismatch = −1


Alignment methods

2-step alignment


Alignment methods

2-step alignment

Step 1: Find candidate positions

• Use read seeds• Hash table-based or Burrows-Wheeler transform-based

heuristic• Balance between speed and accuracy


Alignment methods

2-step alignment

Step 2: Align and report

• Complete alignment with Smith-Waterman• Evaluate alignment(s)


Common issues

Alignment methods

Sequence alignment


Alignment methods

Common issues

Platform specifics

Software


Common issues

Insertions and deletions (indels)


Common issues

Insertions and deletions (indels)

• Local realignment around indels• Per-Base Alignment Qualities (BAQ)


Common issues

Non-unique alignment

How to report non-unique alignments?


Common issues

Non-unique alignment

How to report non-unique alignments?

• Discard entirely

• Choose one randomly

• Report all

• with best quality• above some quality

Depends on the tool


Common issues

Structural variation

• Chromosomal relocation

• Inversion

• Large indels

• Copy-number variation

Use specialized tools


Common issues

Split-read mapping

• Allow aligned read to be split• For example RNA reads on DNA reference


Common issues

Circular alignment

• Circular genome (e.g. bacteria, mitochondria)


Common issues

Circular alignment

• Circular genome (e.g. bacteria, mitochondria)• Most aligners assume linear reference


Common issues

Circular alignment

• Circular genome (e.g. bacteria, mitochondria)• Most aligners assume linear reference• Trick: extend reference


Common issues

Circular alignment


• copy first N bases to the end


Common issues

Circular alignment


• copy first N bases to the end• restore alignment to original reference


Platform specifics

Alignment methods

Sequence alignment


Alignment methods

Common issues

Platform specifics

Software


Platform specifics

Paired-end sequencing


Platform specifics

Paired-end sequencing

• Align reads separately• Choose from non-unique alignments based on pairing


Platform specifics

Color-space (or SOLiD) reads

• Used by 454, Solexa, SOLiD systems• Di-nucleotide encoding• Needs support from alignment software


Platform specifics

Color-space (or SOLiD) reads

Decoding


Platform specifics

Error profile

• Homopolymers• CG-content• Positional (example shown)


Software

Alignment methods

Sequence alignment


Alignment methods

Common issues

Platform specifics

Software


Software

Some popular aligners for NGS

Hash table-based

• Eland• MAQ


Software


Hash table-based

• Eland• MAQ

Burrows-Wheeler Transform-based

• Bowtie• BWA


Software


Hash table-based

• Eland• MAQ

Burrows-Wheeler Transform-based

• Bowtie• BWA

Split-read alignment

• Tophat• GSNAP• Mosaik


Software

Viewers

• IGV, Savant, Geneyous, Tablet


Software

Viewers

• IGV, Savant, Geneyous, Tablet• tview (console-based)


Software

Viewers

• IGV, Savant, Geneyous, Tablet• tview (console-based)• UCSC Genome Browser, GBrowse (web-based)


Questions?

Acknowledgements:

Jeroen Laros

Bas E. Dutilh


Questions?

Image sources

cbsu.tc.cornell.edu/ngw2010/day2 lecture1.pdf

en.wikipedia.org/wiki/Sequence alignment

en.wikipedia.org/wiki/Multiple sequence alignment

www.pitt.edu/ mcs2/teaching/biocomp/tutorials/global.html

www.biology-direct.com/content/4/1/30/figure/F3?highres=y

www.genomesunzipped.org/2012/04/guest-post-accurate-identification-of-rna-editing-sites-from-high

-throughput-sequencing-data.php

www.eplantscience.com/botanical biotechnology biology chemistry/biotechnology/genes genetic

engineering/genes nature concept and synthesis/biotech physical nature dna.php

www.pnas.org/content/109/4/1347/F1.expansion.html

omega.rc.unesp.br/mauricio/curso/bibliografia/22/362/Dibase%20Sequencing%20and%20Color%20Space

%20Analysis.pdf

cgrlucb.wikispaces.com/SAMtoolsSpring2012

and some of my own


martijn vermaat department of human genetics center for ......alignment methods sequence alignment...

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