============================================================
6. Annotating transcript families
============================================================

You can start with the 'trinity-nematostella.renamed.fa.gz' file from the
previous page (:doc:`5-building-transcript-families`) _or_ download
a precomputed one::

      cd /mnt
      curl -O http://public.ged.msu.edu.s3.amazonaws.com/trinity-nematostella.renamed.fa.gz

.. note::

   The BLASTs below will take a *long* time, like 24-36 hours.  If you
   want to work with canned BLASTs, do::

      cd /mnt
      curl -O http://public.ged.msu.edu.s3.amazonaws.com/nema.x.mouse.gz
      curl -O http://public.ged.msu.edu.s3.amazonaws.com/mouse.x.nema.gz
      gunzip nema.x.mouse.gz
      gunzip mouse.x.nema.gz

   However, if you built your own transcript families, you'll need to
   rerun these BLASTs.

Install BLAST
=============

Make sure you've updated BLAST, as in :doc:`installing-blastkit`::

   cd /root

   curl -O ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.24/blast-2.2.24-x64-linux.tar.gz
   tar xzf blast-2.2.24-x64-linux.tar.gz
   cp blast-2.2.24/bin/* /usr/local/bin
   cp -r blast-2.2.24/data /usr/local/blast-data

Doing a preliminary annotation against mouse
============================================

Now let's assign putative homology & orthology to these transcripts, by
doing BLASTs & reciprocal best hit analysis.  First, uncompress your
transcripts file::

   cd /mnt
   gunzip trinity-nematostella.renamed.fa.gz 

Now, grab the latest mouse RefSeq::

   curl -O ftp://ftp.ncbi.nih.gov/refseq/M_musculus/mRNA_Prot/mouse.protein.faa.gz
   gunzip mouse.protein.faa.gz

Format both as BLAST databases::

   formatdb -i mouse.protein.faa -o T -p T
   formatdb -i trinity-nematostella.renamed.fa -o T -p F

And, now, if you haven't downloaded the canned BLAST data above, run
BLAST in both directions.  Note, this may take ~24 hours or longer;
you probably want to run it in screen::

   blastall -i trinity-nematostella.renamed.fa -d mouse.protein.faa -e 1e-3 -p blastx -o nema.x.mouse -a 8 -v 4 -b 4
   blastall -i mouse.protein.faa -d trinity-nematostella.renamed.fa -e 1e-3 -p tblastn -o mouse.x.nema -a 8 -v 4 -b 4

Assigning names to sequences
============================

Now, calculate putative homology (best BLAST hit) and orthology
(reciprocal best hits)::

   python /usr/local/share/eel-pond/make-uni-best-hits.py nema.x.mouse nema.x.mouse.homol
   python /usr/local/share/eel-pond/make-reciprocal-best-hits.py nema.x.mouse mouse.x.nema nema.x.mouse.ortho

Prepare some of the mouse info::

   python /usr/local/share/eel-pond/make-namedb.py mouse.protein.faa mouse.namedb
   python -m screed.fadbm mouse.protein.faa

And, finally, annotate the sequences::

   python /usr/local/share/eel-pond/annotate-seqs.py trinity-nematostella.renamed.fa nema.x.mouse.ortho nema.x.mouse.homol

After this last, you should see::

   207533 sequences total
   10471 annotated / ortho
   95726 annotated / homol
   17215 annotated / tr
   123412 total annotated

If any of these numbers are zero on the nematostella data, then you
probably need to redo the BLAST.

This will produce a file 'trinity-nematostella.renamed.fa.annot', which
will have sequences that look like this::

   >nematostella.id1.tr115222 h=43% => suppressor of tumorigenicity 7 protein isoform 2 [Mus musculus] 1_of_7_in_tr115222 len=1635 id=1 tr=115222 1_of_7_in_tr115222 len=1635 id=1 tr=115222

I suggest renaming this file to 'nematostella.fa' and using it for
BLASTs (see :doc:`installing-blastkit`). ::

   cp trinity-nematostella.renamed.fa.annot nematostella.fa

The annotate-seqs command will *also* produce two CSV files.  The first,
``trinity-nematostella.renamed.fa.annot.csv``, is small, and contains
sequence names linked to orthology and homology information.  The secnod,
``trinity-nematostella.renamed.fa.annot.large.csv``, is large, and
contains all of the same information as in the first but *also* contains
all of the actual DNA sequence in the last column.  (Some spreadsheet
programs may not be able to open it.)  You can do::

   cp *.csv /root/Dropbox

to copy them locally, if you have set up Dropbox (see:
:doc:`../amazon/installing-dropbox`).

.. mouse foo

Next: :doc:`7-expression-analysis`.