Aidan Budd, EMBL Heidelberg

Multiple Sequence Alignments

Aidan Budd, EMBL Heidelberg

Build a Sequence Alignment

Sequences are usually aligned automaticallyMUSCLE, PRANK, CLUSTAL etc.

Also possible 'manually' using tools such as JalView

Hopefully, these demonstrations will highlight that

Alignment is "trivial" (at one level, at least)only involves putting gap characters in the right places

Aidan Budd, EMBL Heidelberg

Build an Automatic MSA

http://www.ebi.ac.uk/Tools/msa/muscle/

Search Internet for "EBI Muscle"

Aidan Budd, EMBL Heidelberg

Build an Automatic MSA

Copy and paste sequences in FASTA format

Click "Submit"

http://www.embl.de/~seqanal/courses/commonCourseContent/sequences/verySimilarHemoglobins_unaligned.fasta

Aidan Budd, EMBL Heidelberg

Build an Automatic MSA

Wait for result to be returned

Click "Download Alignment File" to reach plain-text version of alignment

Aidan Budd, EMBL Heidelberg

Build an Automatic MSA

Download file or copy-paste text into text editor to store alignment on local computer

View alignment in MSA viewer (e.g. JalView) etc.

Aidan Budd, EMBL Heidelberg

Choosing an MSA tool

•CLUSTALX, MUSCLE, PROBCONSdivergent protein sequences

•NASTmultiple alignment of 16S rRNA genes

•PRANKmultiple alignment of relatively similar DNA sequences in an evolutionary context

•EXPRESSO(3DCoffee)multiple alignment of protein sequences, some of which have 3D structural information

•MAUVE, Enredomultiple alignment of genomes

• and many others...

Different tools designed for different tasks

Aidan Budd, EMBL Heidelberg

Examining MSAs:Recognising Patterns

Aidan Budd, EMBL Heidelberg

•Only one of many possible colouring schemes

•Good at highlighting variation in conservation between

•Designed for red/green colour-blindness

CLUSTALX Colouring Scheme

extract from an alignment of p53 proteins

Aidan Budd, EMBL Heidelberg

Amino acids with similar properties drawn with the same colour

extract from an alignment of p53 proteins

e.g. basic residues arginine (R) and lysine (K)

CLUSTALX Colouring Scheme

Aidan Budd, EMBL Heidelbergextract from an alignment of p53 proteins

Residues only coloured...

e.g. lysine (K) in columns with:only "a few" other basic residues (uncoloured)

"many" other basic residues (coloured)

... if some proportion of residues in the column have the same property

CLUSTALX Colouring Scheme

Aidan Budd, EMBL Heidelberg

Hydrophobic: L V I M F W A CPolar: N T S Q

Acidic: D E

Basic: K R Secondary-structure breaking: G P

Large Aromatic Polar: H Y

(CLUSTALX help file fully describes the default colouring rules)

CLUSTALX Colouring Scheme

Aidan Budd, EMBL Heidelberg

http://tardis.nibio.go.jp/cgi-bin/homstrad/showpage.cgi?family=response_reg&disp=str

Response regulator receiver domain

Common Patterns - Buried Beta-Strand

Aidan Budd, EMBL Heidelberg

http://tardis.nibio.go.jp/cgi-bin/homstrad/showpage.cgi?family=response_reg&disp=str

Response regulator receiver domain

Common Patterns - Amphipathic Partially-Buried Alpha-Helices

Aidan Budd, EMBL Heidelberg

ubiquitin conjugating enzyme

Common Patterns - Amphipathic Beta Strands

Aidan Budd, EMBL Heidelberg

Different, more strongly biased (from equal representation of each of the 20 amino acids), sequence composition

Sometimes more variable sequence•more substitutions•more gapsthan globular/structured regions)

Common Patterns - Non-Globular Sequence

Aidan Budd, EMBL Heidelberg

KK

Identifying Mis-Aligned Regions

Identify a region of a sequence that you think is misaligned

Decide how you would "fix" this misalignment

Look at patterns of conservation, and sequences which

Aidan Budd,EMBL Heidelberg

Unusual Sequences: Examples

With CLUSTALX “”Quality”->”Show Low-Scorring Segments” switched on

Short/fragmented sequences

Unusual pattern of "conservation"

Aidan Budd, EMBL Heidelberg

Using MSAs to Improve Prediction of Linear Motifs

Demonstration and Exercise