SAIs in ARB

Simon Locus (Termini_F04/R22) is from Position 1000 to 9962

Dorota Locus is from Position 28425 to end to alignment

SAIs created and currrently saved in MasterDB_95A_mac15Jun.arb in the ARB desktop folder

Phylogenetic placement of short reads

Today I’ve been exploring how to place our short pyrosequencing reads into an evolutionary context.

RAxML EPA webserver ( http://i12k-exelixis3.informatik.tu-muenchen.de/raxml )

  • Downloaded the SILVA 102 release non-redundant reference alignment, but ran into problems with guide tree uploading.  Apparently I need a tree with branch information AND alignment info, which I’m not sure how to do in ARB.  Have e-mailed Alexis and Simon Berger to see if they can help me prepare this file.

Placing sequences onto a tree in ARB

  • I’ve exported my ‘guide tree’ of nematode sequences from my thesis–file is saved as SSU_NematodeThesisRef_w99As_18Aug.arb on the IOMEGA data hard drive.  Full-length reference sequences are labelled as ‘Thesis Ref’ under the import field.  Nematode sequences from the 99A dataset are lablled as ‘Nema99A’ under the import field.

Cox1 Treebuilding and SSU Alignment Edits

Cox1 Tree building—23 May 09

Note: in previous Cox1 treebuilding (files from 4th-11th May), the correct Cox1 alignment to be used is named ‘Align_4May_nts_truncnames

 Today I imported all the cox1 sequences I could find from genbank—the full list of species and accession numbers are listed in the file ‘Cox1_Genbank_Acc_nos’.  The files I worked on are as follows:

  • Genbank_cox1nems.mas (protein sequence alignments—you CANNOT back translate this file to nts in MEGA for some reason)
  • Genbank_cox1nems_ntseq.mas (nucleotide sequence alignment—all sequences had to be re-downloaded from Genbank)
  • Cox1_GBplusEnop_23May.mas (combines all my Enoplid sequences with all the Genbank files I downloaded)
  • Cox1_GBplusEnop_23Maya.mas (removed the following bad sequences: BUS 4, JCC 59, LCL 3, OUS 14, TCR 69, AND WUS 3)
  • Cox1_GBplusEnop_23Mayc.mas (added more Pellioditis sequences from Genbank, and also deleted more sequences as follows: B. ruftipennis, S. lupi, H. muscae, S. sp, R sp., P. sp., C. nassatus, T. skrjabini, G. binucleatum [2 seqs], T. native, C. briggsae, R. iyengari.  These sequences were either too short or looked too divergent to be fitted into the alignment.)
  • Cox1_GBplusEnop_23Mayd.mas (additionally removed the following taxa, which were a bit divergent: O. volvulus, D. immitis, B. malayi, R. lichstensfelsi, B. debrae)

I ran likelihood analyses in PhyML using the divergent and tight alignments (23Mayc and 23Mayd, respectively). I first aligned the translated protein sequences in each .mas file using CLUSTAL in MEGA (default parameters).  The output nucleotide alignments are saved as 23Mayc_DNAaln and 23Mayd_DNAaln.

Alignments had to be saved as sequential PHYLIP files to be run through PhyML—alterted parameters were as follows: HKY model, Ts/Tv ratio estimated, invariable sites estimated, 4 categories of nt substitution, sequential input sequences.

 

Nematode groups edited in the ARB alignment on 22 May 08:

  • Trichura
  • Trichinella
  • Bathyeurystomina
  • Enchelidiidae
  • Pareurystomina
  • Thoracostomopsidae
  • Enoploides
  • Calyptronema
  • Oxystomina
  • Thalassoalaimus
  • Litinium

 The species Birgit used in her paper (Meldal 2007, A revised phylogeny of the phylum Nematoda; I searched for all the accession numbers listed in the appendix [saved as ‘Meldal_appendix_acc_nos.pdf’]), were annotated with the phrase ‘Meldal’ under the field ‘publication_doi’.  Two species weren’t in my ARB database: Trigulla aluta and Belondira apitica.  Somehow, though, although Birgit listed 214 species, I only have 211 marked in ARB (minus the two not found, I missed one somewhere?)

SSU Alignment Edits

The following species were edited on May 20th and 21st:

  • Tripyloides
  • Bathylaimus
  • Enoplus
  • Chaetonema
  • Metachromadora
  • Oncholaimus
  • Adoncholaimus
  • Spirinia
  • Viscosia
  • Enoplus
  • Enoplolaimus
  • Mesacanthion
  • Paramesacanthion
  • Sabatieria
  • Daptonema
  • [Mesodorylaimus]
  • [Leptonchus (4:0)]
  • [Bathyeurystomina]

After two days of edits, I rebuilt the entire NJ tree  at 5pm on May 21st.

(Bold genera names mean I search and queried all sequences with that genera name. [Bracket] genera names mean these genera were edited alongside Bold genera as reference sequences–thus, not all sequences with this genera assignment have been searched and edited):