Coursework for UCB BioE 231 - Lab 2

This lab analyzes and plots the data given in the sequence file seq.fa

Command

A step by step explanation of Lab 2

Generating a phylogenetic tree

To align multiple sequences and generate multiple sequence alignment (MSA), we used the command:

be231-13@jitterbug ~/lab2 [5:21pm]> muscle -in seqss.fa -out seqss.aligned.fa

The result:

MUSCLE v3.8.31 by Robert C. Edgar

http://www.drive5.com/muscle
This software is donated to the public domain.
Please cite: Edgar, R.C. Nucleic Acids Res 32(5), 1792-97.

seqss 48 seqs, max length 2217, avg  length 2209
00:00:00     11 MB(1%)  Iter   1  100.00%  K-mer dist pass 1
00:00:00     11 MB(1%)  Iter   1  100.00%  K-mer dist pass 2
00:00:07     83 MB(5%)  Iter   1  100.00%  Align node
00:00:07     83 MB(5%)  Iter   1  100.00%  Root alignment
00:00:11     83 MB(5%)  Iter   2  100.00%  Refine tree
00:00:11     83 MB(5%)  Iter   2  100.00%  Root alignment
00:00:11     83 MB(5%)  Iter   2  100.00%  Root alignment
00:00:26     83 MB(5%)  Iter   3  100.00%  Refine biparts
00:00:42     83 MB(5%)  Iter   4  100.00%  Refine biparts
00:00:42     83 MB(5%)  Iter   5  100.00%  Refine biparts
00:00:42     83 MB(5%)  Iter   5  100.00%  Refine biparts

Comparison of input and output of the command:

The seqs.fa file The seqs.aligned.fa file

After this, we utilized Morgan Price’s excellent tool FastTree turn multiple alignment into a Newick-formatted tree with the following command:

be231-13@jitterbug ~/lab2 [5:41pm]> FastTree -nt < seqss.aligned.fa > trees.nwk

The result:

FastTree Version 2.1.10 SSE3
Alignment: standard input
Nucleotide distances: Jukes-Cantor Joins: balanced Support: SH-like 1000
Search: Normal +NNI +SPR (2 rounds range 10) +ML-NNI opt-each=1
TopHits: 1.00*sqrtN close=default refresh=0.80
ML Model: Jukes-Cantor, CAT approximation with 20 rate categories
Initial topology in 0.07 seconds
Refining topology: 22 rounds ME-NNIs, 2 rounds ME-SPRs, 11 rounds ML-NNIs
Total branch-length 0.954 after 0.87 sec2, 1 of 46 splits
ML-NNI round 1: LogLk = -15621.453 NNIs 8 max delta 16.59 Time 1.37
Switched to using 20 rate categories (CAT approximation)20 of 20
Rate categories were divided by 0.740 so that average rate = 1.0
CAT-based log-likelihoods may not be comparable across runs
Use -gamma for approximate but comparable Gamma(20) log-likelihoods
ML-NNI round 2: LogLk = -14251.186 NNIs 2 max delta 0.00 Time 1.69
Turning off heuristics for final round of ML NNIs (converged)
ML-NNI round 3: LogLk = -14251.086 NNIs 0 max delta 0.00 Time 2.04 (final)
Optimize all lengths: LogLk = -14251.086 Time 2.15
Total time: 2.81 seconds Unique: 48/48 Bad splits: 0/45

The resulting newwick will be drawn in the iPython file.

Identifying sequences by BLAST & Calculating sequences statistics for each cluster

All the details are explained in the iPython file.

Here we show how the tree was clustered.

Cluster 1
hu.31
hu.32
hu.14
Cluster 2
hu.44
hu.46
hu.43
hu.48
Cluster 3
pi.3
pi.1
pi.2
Cluster 4
rh.43
Cluster 5
rh.58
rh.57
hu.39
rh.49
rh.51
rh.61
rh.52
rh.50
rh.53
rh.64
Cluster 6
bb.1
bb.2
rh.10
hu.17
hu.6
Cluster 7
rh.2
rh.40
hu.41
rh.38
hu.66
hu.67
hu.42
hu.37
hu.40
Cluster 8
cy.2
rh.54
rh.55
rh.48
rh.62
Cluster 9
rh.35
rh.36
rh.37
Cluster 10
cy.4
cy.6
cy.3
cy.5
rh.13

The cluster representatives are the first specimen of each cluster in the list. Their BLAST analysis reveals that:

• The hu.31 sequence corresponds to the Adeno-associated virus isolate hu.31 capsid protein VP1 (cap) gene, complete dds.
• The hu.44 sequence corresponds to the Adeno-associated virus isolate hu.44 capsid protein VP1 (cap) gene, complete cds.
• The pi.3 sequence corresponds to the Adeno-associated virus isolate pi.3 capsid protein VP1 (cap) gene, complete cds.
• The rh.58 sequence corresponds to the Adeno-associated virus isolate rh.58 capsid protein VP1 (cap) gene, complete cds.
• The rh.58 sequence corresponds to the Adeno-associated virus isolate rh.58 capsid protein VP1 (cap) gene, complete cds.
• The bb.1 sequence corresponds to the Non-human primate Adeno-associated virus isolate AAVbb.1 capsid protein (VP1) gene, complete cds.
• The rh.2 sequence corresponds to the Non-human primate Adeno-associated virus isolate AAVrh.2 capsid protein (VP1) gene, complete cds.
• The cy.2 sequence corresponds to the Non-human primate Adeno-associated virus isolate AAVcy.2 capsid protein (VP1) gene, complete cds.
• The rh.35 sequence corresponds to the Non-human primate Adeno-associated virus isolate AAVrh.35 capsid protein (VP1) gene, complete cds.
• The cy.4 sequence corresponds to the Non-human primate Adeno-associated virus isolate AAVcy.4 capsid protein (VP1) gene, complete cds.

Built With

• Matplotlib - Python 2D plotting library
• BioPhython - a set of freely available tools for biological computation

Authors

• Thomas Galeon - Initial work - TheGaga
• Zach Lyu - Initial work - bijiuni