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T-ACE: Transcriptome Analysis and Comparison Explorer

Background

T-ACE was developed for the analysis and organization of transcriptome projects but is also helpful for the organization of small sequence datasets e.g. extracts of a large transcriptome databases. T-ACE does not provide an assembly function, thus data gained by NGS projects (e.g. 454, Illumina, SoliD) have to be assembled prior to the upload (e.g. using Newbler, Celera or TGICL). For transcriptome comparisons T-ACE was designed to work with a dataset composed of several transcriptomes of different treatments or tissues, which are assembled into a consensus transcriptome. Subsequently reads of the different transcriptomes are again mapped against the consensus transcriptome for information of transcriptome expression pattern i.e. number of reads per specific contig.

Setting up a T-­ACE Client

If there is already a running T-ACE database server, there is not much to do to set up a T-ACE client. At first TCL/TK 8.5.10.1 or higher should be installed. The easiest way to do this is to use an ActiveState package; it contains already most necessary additional TCL packages. The additional packages are listed in the Required Software section below.
When TCL is installed, just download T-ACE from this website and extract it to any convenient directory. Then just double-click on the T-ACE.tcl file in the T-ACE folder and the client interface should open.
To set up the connection to the T-ACE database server open the 'Database connection' in the config menu.
It is also recommended to install additional software like NCBI-Blast+, Phobos and Primer3; otherwise some T-ACE features will not be working. Look in T-ACE manual or the Additional software section below for specifications.
 

Setting up a T­ACE database server

To set up a T-ACE database server a PostgreSQL server is necessary. The additional software pgAdmin3 should also be installed for an easier handling of the Postgres database. For the functions used in T-ACE parent database the additional module 'postgresql-pltcl-8.3' is needed.
After the installation of the PostgreSQL server some configurations have to be made:

Resetting the password of the postgresuser:

sudo su postgres -c psql template1
template1=# ALTER USER postgres WITH PASSWORD 'password';
template1=# \q

Then it is necessary to change some lines in the 'postgresql.conf':

#listen_addresses = 'localhost'
to
listen_addresses = '*'

#password_encryption = on
to
password_encryption = on

The 'pg_hba.conf' file should be edited to use md5 for password checks.
If T-ACEpg is used for non-local access the following line should be added:

# IPv6 local connections:

This would allow the user 'tace_user' to access the database 'tace_parentdb' from anywhere in the internet. Change the CIDR-ADDRESS to limit the access further.
The normal T-ACE version accesses the Postgres database via a php script; this means it is not necessary to make the Postgres server accessible for external IP-addresses.

Now the server has to be restarted:

sudo /etc/init.d/postgresql-8.4 restart


Then an Apache web server is needed to allow external T-ACE clients the access to the Postgres database. An external T-ACE client will access the Postgres database through a php script which runs on the Apache server, so make sure that php scripts can be executed correctly on the server.
Now TCL/TK and T-ACE can be installed. For configuring a 'tace_parentdb' the additional TCL package 'Pgtcl' is needed, otherwise the T-ACE_DB_manager cannot be used.
When T-ACE is extracted, copy the T-ACE.php into the /htdocs folder of the Apache web server. To set up the T-ACE parent database, functions and login roles follow the instructions in the T- ACE_DB_manager. When this is done the first T-ACE transcriptome database can be created, for this look here.
Additionally wwwblast should be installed on an apache server, but it does not need to run on the same machine as the Postgres server. Then blast databases of the T-ACE databases have to be copied to the /db directory of blast server and added to the blast.rc file, otherwise the blast function of T- ACE will not work.
 

Downloads:

T-ACE software

T-ACE references

T-ACE reference sql files for upload to the T-ACE parent database.
The files contain data for the respective refdb schema table. For example the 'T-ACE_refDB_blastprodom.sql' contains the prodom IDs and descriptions, the 'T-ACE_refDB_interpro2go.sql' contains the interpro to GO relations.
The 'T-ACE_refDB_all.sql' contains data of all other reference files in this list. If you download the 'T-ACE_refDB_all.sql' you don't have to download any other reference file.

The reference files on this page won't contain always the newest reference lists, so you may use the T-ACE_DB_manager to upload the newest reference files. In the T-ACE manual you will find links to the reference download webpages.
To upload a sql to the PostgreSQL server file extract the file and use:

• psql -h [server host] -U [user name] [name of T-ACE parentDB] < [name of SQL file].sql

T-ACE Demo

To test T-ACE it is not necessary to set up a PostgreSQL server and install the T-ACE parent database and the example databases. A webserver is available, where T-ACE can be tested. To test T-ACE only TCL and the T-ACE client have to be installed.
To access the server use the following database connection settings (see 'Config'-menu -> 'Database connection' in the the T-ACE client) or download the 'T-ACE.conf' file below copy it to the T-ACE/conf folder.
See also Tutorial 1.

Database connection:

Server autdentification

Blast parameters:

Login parameters:

If a local installation is preferred, it is necessary to upload the T-ACE_refDB_all.sql to the refdb schema of the T-ACE parent database, otherwise the description of annotations in the example databases will not be available.

T-ACE example databases

Sabella spallanzanii database:

S. spallanzanii individuals were sampled in May 2009 from Moulin Blanc harbor (Brest, France) by staff of the Station Biologique (Roscoff, France), stored in RNAlater and send to the Institute of Clinical Molecular Biology (Kiel, Germany). A single individual was taken from the RNAlater, dissected and the fan tissue removed, blotted dry and frozen in liquid nitrogen for further processing. Frozen tissue was ground in liquid nitrogen and total RNA extracted using the Qiagen RNeasy kit in combination with the QiaShredder from Qiagen (Hilden, Germany). mRNA was purified by using the Qiagen Oligotex mRNA purification kit (Qiagen, Hilden, Germany) and cDNA generated with the SMART cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA, USA). Quality control in each extraction step was investigated using a nanodrop spectrophotometer (Peqlab, Erlangen). Only samples with a 260/280 and 260/230 ratio >1.8 were used for sequencing. Samples were prepared for 454 sequencing according to the manufacturers protocol and sequenced on a Genome Sequencer FLX system (454 Life Sciences, Branford, CT, USA) with FLX and Titanium chemistry. The resulting 454 sequences were extracted from FLX output files using the 'sffinfo' script from Roche.

Mytilus galloprovincialis database:

The generation of M. galloprovincialis sequences are described in detail in Craft et al.. Original sequences were downloaded in December 2010 from the MG-RAST portal (https://metagenomics.nmpdr.org) and cleaned and assembled as described in Philipp et al (submitted). In total 104123 MG-RAST sequences (av. length 211bp) of M. galloprovincialis were assembled into 12827 contigs (av. length 279bp) and 40972 singletons (av. length 207bp). Using AMOS the different reads originating from either gill, foot, digestive gland or mantle tissue were mapped against the generated contigs and the Mytilus edulis mitochondrion genome (GI:55977238), which resulted in the final contigs and assigned reads from the different tissues.

Annotation of M. galloprovincialis and S. spallanzanii contigs:

Putative gene names and protein domains were assigned to all contigs by using the BLASTX algorithm against the UniprotKBSwissprot and UniRef100 protein databases of UniProt Knowledgebase (UniProtKB, https://www.expasy.org/sprot/) with a cut off e-value of e <= 10^-3, as well as tBLASTx (e <= 10^-3) and BLASTn (e <= 10^-10) against the NCBI non-redundant nucleotide (nt) database (https://www.ncbi.nlm.nih.gov). The Sabella dataset was also investigated for conserved domains, by running the assembled contigs via InterProScan. Gene Ontology (GO) terms were deduced from BLAST and InterProScan results.

References: