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LSM2241AY0910 Semester 2

MiniProject Briefing

Round 5

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FOCUS

Identify and analyze sequence differences among NF-κB protein sequences

Specific objectives

1. Analyze mammalian NF-κB proteins and their homologues

2. Generate sequence patterns for conserved regions in mammalian NF-κB proteins and their homologues

3. Map conserved sequence patterns to 3D structures

Topics for Round 5

• Data collection - DONE

• Data processing - DONE

• Data analysis– Phylogenetic analysis

• Multiple Sequence Alignment - DONE• Generating a phylogenetic tree - DONE

– Sequence pattern analysis – TODAY– Structural analysis

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Round 4 Review

• Adding root sequence to your MSA

• Editing your MSA

• Generating a phylogenetic tree with bootstrap values

• Any questions?

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Sequence Pattern Analysis

Goal

To identify patterns within conserved domains of NF-κB proteins

How?

Generate Prosite patterns within the conserved domains of selected mammalian NF-κB proteins using the PRATT tool

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Identifying highly conserved sequences from your phylogenetic tree

• From the phylogenetic tree generated in the previous section, record the accession numbers of the sequences that are present in each statistically significant clade (bootstrap value ≥ 70)

Note: Record the accession numbers for each clade separately

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Identifying highly conserved sequences from your phylogenetic tree

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Identifying highly conserved sequences from your phylogenetic tree

• Extract the aligned conserved domain sequences for each statistically significant clade from the edited multiple sequence alignment file

• Launch BioEdit: Edit Search Search for titles that contain a list of substrings

• In the popup window, type or paste the list of accession numbers you want to extract in the blank field and click “OK”

Note: Do this separately for each statistically significant clade

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Identifying highly conserved sequences from your phylogenetic tree

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Identifying highly conserved sequences from your phylogenetic tree

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Identifying highly conserved sequences from your phylogenetic tree

• Now, all the sequences containing the accession numbers you have keyed in will be selected.

• Copy these sequences by selecting Edit Copy Sequence(s).

• Open a blank alignment file (File New Alignment) and paste your sequences into the blank alignment file (Edit Paste)

• Save your new alignment in FASTA format • Repeat for sequences in each statistically significant clade

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Generating Patterns

• Do you think you need to do some processing to the alignment of each clade before generating a pattern?

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Original sequence

Clade alignment

new slide

Generating Prosite patterns

• Use the PRATT tool to generate Prosite patterns for parts of each statistically significant clade (follow the instructions in P7)

• Scan your patterns against all mammalian NF-κB sequences– How do you obtain these sequences?

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slide updated

Generating Prosite patterns

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Copy and paste the contents of nfkb_all.fas in the ScanProsite page, under “Sequence(s) to be scanned”

Generating Prosite patterns

• Analyze your results:– Compare and contrast each Prosite pattern. How are

they similar or different? – Compare the results for each Prosite pattern. How

many hits do you get for each Prosite pattern?– Which are the proteins that contain your pattern? – How accurate is your pattern? – Are your results reliable? If not, what can you do to

generate more reliable results?

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Generating Prosite patterns

• Repeat the ScanProsite step again

• This time, scan your patterns against UniprotKB/Swiss-Prot, UniProtKB/TrEMBL and PDB. Select the option: include all splice variants.

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Generating Prosite patterns

• Compare and contrast your results– How many hits do you get now? – Do you get the same results as the last step? Why?

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Generating Prosite patterns

• Analyze your results:– Compare and contrast each Prosite pattern. How are

they similar or different? – Compare the results for each Prosite pattern. How

many hits do you get for each Prosite pattern?– Which are the proteins that contain your pattern? – How accurate is your pattern? – Are your results reliable? If not, what can you do to

generate more reliable results?

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Generating Prosite patterns

• Points for discussion– What is the significance of sequence pattern

analysis? In what ways are Prosite patterns useful?

– What is the significance of your results? – Are there any other pattern generation software?

Do they generate better results?– How can you improve on your results?

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Topics for Round 5

• Data collection - DONE

• Data processing - DONE

• Data analysis– Phylogenetic analysis

• Multiple Sequence Alignment - DONE• Generating a phylogenetic tree - DONE

– Sequence pattern analysis - DONE– Structural analysis – NEXT WEEK

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