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Comparative Microbial Genomics - Exercise 3
Thursday, 4. October 2007
Today your task will be to explore conservation in bacterial ribosomal RNA by generating multiple alignments.
Based on sequence alignment you will be able to derive sequence similarity and later to draw phylogenetic trees
to compare bacterial genomes.
A current research project in the Comparative Microbial genomics group deals with accurate and fast prediction of rRNA genes. This exerciese will
briefly demonstrate using the results from the prediction tool - RNAmmer.
The software we will be using today is downloadable from these two places:
ClustalX
TreeView
BACKGROUND
Most other methods for predicting rRNA uses BLAST. The disadvantage of BLAST is that it is a linear method that weights each position in the alignment equally. However not every position of the rRNA molecule is equally conserved and RNAmmer
therefor use profile hidden markov models which are build from structural alignments of thousands of rRNA sequences. These models will recognize conserved
regions and weight each conserved loci differently. To learn more about profile HMMs, visit hmmer.wustl.edu
Step 1 - Getting rRNA
Your task is here to access first the CBS webpages and getting 16S sequences predicted by RNAmmer for the genomes listed below.
CBS
| Name | Project ID | Accession |
|---|
| Bacillus halodurans C-125 | 235 | BA000004 |
| Bacillus licheniformis ATCC 14580 | 12388 | CP000002 |
| Bacillus licheniformis ATCC 14580_DSM | 13082 | AE017333 |
| Candidatus Carsonella ruddii PV | 17977 | AP009180 |
| Escherichia coli K12 | 225 | U00096 |
| Escherichia coli CFT073 | 313 | AE014075 |
| Haemophilus influenzae Rd KW20 | 219 | L42023 |
| Mannheimia succiniciproducens MBEL55E | 13068 | AE016827 |
| Pasteurella multocida Pm70 | 39 | AE004439 |
Add the sequence that you find here:
>16s_A
AGGTGATCCAACCGCAGGTTCCCCTACGGTT
ACCTTGTTACGACTTCACCCCAGTCATGAATCATACCGTGGTAAACGCCCCCCCGAAGGT
TAAGCTATCTACTTCTGGTACAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGC
CCGGGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTCCGACTTCATGG
AGTCGAGTTGCAGACTCCAATCCGGACTACGATGCACTTTCTGAGATTCGCTCTACCTCG
CGGTATCGCCGCCCTCTGTATGCACCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGGC
CATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTTGAG
TTCCCATCTTTACATGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACC
CAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGCTCCCGAAG
GCACTCCCGTATCTCTACAGGATTCTCTGGATGTCAAGAGTAGGTAAGGTTCTTCGCGTT
GCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTT
TTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTATCACGTTAGCTACGAGCACCAAG
CCTAAAGCCCAATCCCCAAATCGACAGCGTTTACCGCGTGGACTACCAGGGTATCTAATC
CTGTTTGCTCCCCACGCTTTCGCATATGAGCGTCAGTATCTTCCCAAGGGGCTGCCTTCG
CCTTCGGTATTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCTACCCCTC
CCTAAAGTACTCTAGACTCCCAGTCTGAAATGCTATTCCCAGGTTAAGCCCGGGGCTTTC
ACATCTCACTTAAAAGTCCGCCTGCATGCCCTTTACGCCCAGTTATTCCGATTAACGCTC
GCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGTGATTA
ACGTCAATTGCTTGTCCTATTAAAACAAACACCTTCCTCGTCACCGAAAGAACTTTACAA
CCCGAAGGCCTTCTTCATTCACGCGGCATGGCTGCATCAGGGTTCCCCCCATTGTGCAAT
ATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGT
CATCCTCTCAGACCAGCTAGAGATCGTCGGCTTGGTAGGCCTTTACCCCACCAACTACCT
AATCCCACTTGGGCTCATCTTATGGCGGGTGGCCCGTAAGTCCCACCCTTTAATCCGAAG
ATATTACGCGGTATTAGCGACAGTTTCCCGTCGTTATCCCCCTCCATAAGCCAGATCCCC
AAGCATTACTCACCCGTCCGCCACTCGTCGGCAAAGAAAGCAAGCTTTCTTCCCGCTACC
GTTCGACTTGCATGTGTTAAGCCTGCCGCCAGCGTTCAATCTGAGCCATGATCAAACTCT
Step 2: Produce a multiple alignment using clustalx
Step 3:Examine the clustal alignment. Does the alignment look acceptable?
Is there something strange with any of the sequences?
Step 4:In clustalx produce a tree by clicking Trees > Draw N-J tree (neighbor joining tree) and click ok (the tree is saved in phylip format) - press OK
Step 5: Examine the tree using TreeView. Are some of the sequences misplaced?
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