SequenceAnalysis:MultipleSequenceAlignment(MSA)andPhylogeny ExerciseSheet3 SoftwarewerkzeugederBioinformatik

Softwarewerkzeuge der Bioinformatik

Prof. Dr. Volkhard Helms

PD Dr. Michael Hutter, Markus Hollander, Marie Detzler

Winter Semester 2020/2021

Saarland University Center for Bioinformatics

Exercise Sheet 3

Sequence Analysis: Multiple Sequence Alignment (MSA) and Phylogeny

Learning objective: The goal is to learn how to generate multiple sequence alignments, how to interpret them e.g. regarding sequence conservation and their usefulness for different types of questions. Additionally, you are going to apply the Sankoff algorithm and learn how to work with phylogenetic trees.

Exercise 3.1: Homologous sequences, conserved domains and phylogenetic trees Tools for generating multiple alignments: http://www.ebi.ac.uk/Tools/msa

a) Save the sequence of proteinQ38856together with 9 homologous sequences in multi–fasta–

format.

b) Find highly conserved parts of the sequences with a tool of your choice.

c) Do all amino acids have to be highly conserved in order to conclude that the proteins are homologous?

d) Let’s assume that you want to locate the active centre of a protein but only have the protein sequence without the corresponding structure. How can a multiple sequence alignment help you to solve this problem?

e) Generate a multiple sequence alignt of 50 homologous sequences with the same tool.

f) What differences do you observe between the two alignments?

g) Look at the phylogenetic tree of the sequences in 3.1.e) and find three biological groups (plants, fungi and animals).

Exercise 3.2: Comparison of various tools

The following multiple sequence alignments were generated with different tools:

Tool Protein Alignment

ClustalW

FOS Rat MMF S GFNADYEAS S SRCSSASPAGDSL SYYHSPADSF S SMGS PVNTQDFC MMF S GFNADYEAS S SRCSSASPAGDSL SYYHSPADSF S SMGS PVNTQDFC MMYQGFAGEYEAP S SRCSSASPAGDSLTYYPSPADSF S SMGS PVNSQDFC – MFQAFPGDYDS – GSRCSS– SP S AESQ – –YLSSVDSFGS P PTAAASQE –C – MFQAFPGDYDS – GSRCSS– SP S AESQ – –YLSSVDSFGS P PTAAASQE –C

* : . . * . : * : : . ** * ** * * : . : * * * . . * * * . * : .. : * : * FOS MOU

FOS CHIC FOSB MOU FOSB HU

MAFFT

FOS Rat MMF S GFNADYEAS S SRCSSASPAGDSLS YYHSPADSF S SMGS PVNTQDFC MMF S GFNADYEAS S SRCSSASPAGDSLS YYHSPADSF S SMGS PVNTQDFC MMYQGFAGEYEAP S SRCSSASPAGDSLTYYPSPADSF S SMGS PVNSQDFC – MFQAFPGDYD– SGSRCSS– SP S AES– – QYLSSVDSFGS P PTAAASQE –C – MFQAFPGDYD– SGSRCSS– SP S AES– – QYLSSVDSFGS P PTAAASQE –C

* : . . * . : * : . . ** * ** * * : . : * * * . . * * * . * : . . : * : * FOS MOU

FOS CHIC FOSB MOU FOSB HU

MUSCLE

FOS Rat MMF S GFNADYEAS S SRCSSASPAGDSL SYYHSPADSF S SMGS PVNTQDFC MMF S GFNADYEAS S SRCSSASPAGDSL SYYHSPADSF S SMGS PVNTQDFC MMYQGFAGEYEAP S SRCSSASPAGDSLTYYPSPADSF S SMGS PVNSQDFC – MFQAFPGDYD– SGSRCSS– SP S AESQ – –YLSSVDSFGS P PTAAASQE –C – MFQAFPGDYD– SGSRCSS– SP S AESQ – –YLSSVDSFGS P PTAAASQE –C

* : . . * . : * : . . ** * ** * * : . : * * * . . * * * . * : .. : * : * FOS MOU

FOS CHIC FOSB MOU FOSB HU

Clustal Omega

FOS Rat MMF S GFNADYEA S SSRCSSASPAGDSL S YYHSPADSF S SMGS PVNTQDFC MMF S GFNADYEA S SSRCSSASPAGDSL S YYHSPADSF S SMGS PVNTQDFC MMYQGFAGEYEAPSSRCSSASPAGDSLTYYPSPADSF S SMGS PVN S QDFC – MFQAFPGDYDS GS–RCSSS PSA – – –ESQYLSSVDSFGS P PTA– AA S QEC – MFQAFPGDYDS GS–RCSSS PSA – – –ESQYLSSVDSFGS P PTA– AA S QEC

* : . . * . : * : : * * * **: * : * * . * * * . * : : . : * FOS MOU

FOS CHIC FOSB MOU FOSB HU

Compare the MSAs.

a) Are there differences regarding the gap arrangement?

b) Does this change the degree of conservation in the coloured columns?

Exercise 3.3: Conserved motifs

Use Clustal Omega to generate a multiple sequence alignment of the sequences provided on the lecture website (sequences1.fasta). Locate uninterrupted, highly conserved areas of at least length 10 and save them as potential motifs for exercise sheet 4 (based on FOSB MOUSE).

Exercise 3.4: Outgroup

a) Generate an MSA of the sequences provided on the lecture website (sequences2.fasta).

b) Is everything conserved?

c) Which species differs from the rest?

d) Construct a phylogenetic tree.

Exercise 3.5: Sankoff algorithm

Which base was likely in the ancestor sequence at the given position of the alignment? Use the Sankoff algorithm and the given cost function.

A C G T

G C

A T A C G T

T G C

A A C G T A C G T

r

v10 v11

v7 v8 v9

v1 v2 v3 v4 v5 v6

l₁ l₂ l₃ l₄ l₅ l₆ l₇ l₈ l₉ l₁₀ l₁₁ l₁₂ l₁₃

{G} {A} {T} {A} {C} {T} {G} {A} {T} {A} {A} {G} {G}

A C G T

A C T

A C G T

A C G T

A C G T

A C G T

C G T

−→Base in the ancestor sequence:

Cost function:

A C G T

A 0 2 1 2

C 2 0 2 1

G 1 2 0 2

T 2 1 2 0

Have fun!