This document provides a detailed description of the methods used in the creation of a comprehensive phylogeny for cephalopods with brain volume data available. The analysis uses data provided by Lindgren et al..

All the code is available on Github under a MIT license and may be used to recreate the analysis.

We use the table published by Lindgren et al. and obtain the DNA sequences using the genebank IDs. The constructed Cephalopods are stored as Go structs.

./construct-cephalopods -csv lindgren-paper/Lindgren-et-al-2012-1471-2148-12-129-s2.csv -gob 2015-04-20-cephalopods.gob

Now we split the Cephalopods by Gene, because not every Cephalopod has sequences for all genes used in the analysis (missing data). We use only cephalopods that have at least 4 sequenced genes.

./get-species-subset -gob 2015-04-20-cephalopods.gob -minseq 4
Total number of Cephalopods:  414
Number of valid species:  181
Writing fasta file for gene:  H3
Writing fasta file for gene:  odh
Writing fasta file for gene:  COI
Writing fasta file for gene:  18S
Writing fasta file for gene:  16S
Writing fasta file for gene:  cytb
Writing fasta file for gene:  pax
Writing fasta file for gene:  12S
Writing fasta file for gene:  opsin
Writing fasta file for gene:  28S

Because we have brain data only from a subset of species, we use only those genes that contain those species, namely

12S
16S
18S
COI

The 12S, 16S and 18S sequences are non-coding and we can therefore just use the aligned nucleotide sequences. For the COI sequences, we can produce a codon alignment using TranslatorX (http://translatorx.co.uk/). I modified the perl script so it would use mafft with einsi settings.

Ceph402:topac:537939951 # Excluded, some plant, no brain data
Ceph322:ropal:4003471 # Excluded, sequence degrades at end, no brain data

#### Changed

Ceph269:ocvul:537938871 This is a wrong sequence GID, actually some sort of plant. Added sequence http://www.ncbi.nlm.nih.gov/nuccore/AB052253.1 >Ceph269:ocvul:11611627

# Use MAFFT with EINSI settings (edited perl script)
./translatorx_vLocal.pl -i ../sequences/COI/COI.valid.cleaned.fa -c 5 -o ../sequences/COI/COI.valid.cleaned.aln -t T -p F

# Use standard settings for other genes.
mafft 12S.valid.fa > 12S.valid.aln
mafft 16S.valid.fa > 16S.valid.aln
mafft 18S.valid.fa > 18S.valid.aln

#### Trimming

I trimmed the alignment produced by TranslatorX in order to delete gappy bits

Ceph321:ropac:5353806/1-585 # DELETED beginning

Deleted first and last two codons

Final result stored in COI.valid.cleaned.trimmed.codon.fa and *.valid.aln.

In order to analyse the species, we concatenated the alignments for all genes and added gaps for missing data.

./concatenate-alignments -in sequences/alignments -out 2015-03-28-cephalopods-12S16S18SCOI.aln
[12S 16S 18S COI]
Number of sequences in alignment:  119
Number of sequences in alignment:  171
Number of sequences in alignment:  92
Number of sequences in alignment:  125

# Exabayes partitions
DNA, 12S = 1-669
DNA, 16S = 670-1422
DNA, 18S = 1423-5674
DNA, COI-c1c2 = 5675-6334\3, 5676-6334\3
DNA, COI-c3 = 5677-6334\3

#Change to phylip format
./fasta2phylip -in cephylopods-transfer/2015-04-28-cephalopods-12S16S18SCOI.aln.fa -out cephylopods-transfer/2015-04-28-cephalopods-12S16S18SCOI.aln.phy

Now we run Exabayes with the following command: