mptp_exercise

Commands for installing mptp

$ git clone https://github.com/Pas-Kapli/mptp.git

$ cd mptp

$ ./autogen.sh

$ ./configure

$ make

$ sudo make install

Data pre-processing

1.1. Infer alignment:

$ mafft Anolis.fas > Anolis_mafft.fas

Need to be much more careful with the alignment when using datasets with many indels (e.g. 16S rRNA)

1.2. Remove identical sequences:

$ raxmlHPC-PTHREADS-SSE3 -f c -s Anolis_mafft.fas -m GTRGAMMA -n read_alignment

On the screen and the RAxML_info.read_alignment file there is the following information

Found 13 sequences that are exactly identical to other sequences in the alignment.
Normally they should be excluded from the analysis.```	

A reduced file is automatically created: Anolis_mafft.fas.reduced in phylip format 

Check the difference: 

```$ head -n 1 Anolis_mafft.fas.reduced```

```$ grep ">"```

```grep ">" Anolis_mafft.fas | wc -l ```

_**Q: How many sequences were in the fasta file and how many are there in the reduced phylip file?**_

#### Convert phylip to fasta (we will need it for mptp):

``` $ phylip_to_fasta.py Anolis_mafft.fas.reduced ```

The output file will be named Anolis_mafft.fas.reduced.fasta

### 1.3. Infer phylogenetic tree with RAxML:

``` $ raxmlHPC-PTHREADS-SSE3 -s Anolis_mafft.fas.reduced.fasta -m GTRGAMMA -n Anolis -p $RANDOM -T 2 -o GBGC12094-13_Polychrus ```

_Using the -o argument we retrieve a rooted phylogeny, if not then we can root it with mptp later_

## ``Species'' delimitation with mptp:

Check the mptp options:

```$ mptp --help```

Run delimitation with the multi-rate algorithm

_If the phylogeny is unrooted use the ``--outgroup taxon_name1,taxon_name2'' for rooting the phylogeny prior to the delimitation_

``` $ mptp --ml --multi --tree_file RAxML_bestTree.Anolis --output_file Anolis_multi_delimitation ```

Check the output files:

``` $ firefox Anolis_multi_delimitation.svg ```

``` $ nano Anolis_multi_delimitation.txt ```

Q: How many putative species are delimited?

Detect minimum branch length:

``` $ mptp --tree_file RAxML_bestTree.Anolis --minbr_auto Anolis_mafft.fas.reduced.fasta --output_file minbr ``` 

Re-run delimitation taking the minimum branch length into account:

``` $ mptp --ml --multi --tree_file RAxML_bestTree.Anolis --output_file Anolis_multi_delimitation --minbr 0.0014695325 ```


Re-run but crop the outgroup taxa:

``` $ mptp --ml --single --tree_file RAxML_bestTree.Anolis --output_file signle --outgroup GBGC1209413_Polychrus --outgroup_crop ```

_**Q: Does the delimitation change when by using the minimum branch length or the crop-outgroup command? Why? When would these options matter?**_

Repeat the exercise with the single-rate algorithm:

_**Q: How do the results differ from the multi-rate PTP?**_ 

1.5. Support values:

Perform MCMC sampling (proportional to the likelihood) for the multi and the single rate PTP:
``` $ mptp --tree_file RAxML_bestTree.Anolis --minbr 0.0014695325 --mcmc 10000000 --mcmc_log 1000000 --multi --mcmc_runs 2 --output_file mcmc_multi ```

``` $ firefox mcmc_multi.SOME_NUMBER.combined.svg ```

_**Q: Did the two runs converge?**_
_**Q: Did the runs converge to the Maximum Likelihood solution?**_