Transcriptomic GC content Analysis (TGCA)
Version 1 (2017)
Tutorial
INDEX
REQUIREMENTS
SET UP
1. Preparation of expression data
1.1. Export of expression data from TRAM
1.2. Import
of the expression data in TGCA
2. Preparation of sequences
2.1. Export of sequences from 5'_ORF_Extender
2.2. Importation of sequences in TGCA
3. Description of TGCA Table
GENERAL DEFINITIONS
4.1 File
4.2 Table
4.3 Record
4.4 Field
4.5 Layout
4.6 Browse Mode
4.7 Find Mode
4.8 Preview Mode
MENU AND COMMANDS
5.1 TGCA
5.2 File
5.3 Edit
5.4 View
5.5 Records
5.6 Scripts
5.7 Help
TROUBLESHOOTING
REQUIREMENTS
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The minimum
software requirements
are:
Mac OS X 10.6, OS X
Lion 10.7, OS X Mountain Lion 10.8;
Windows XP
Professional, Home Edition (Service Pack 3);
Windows Vista
Ultimate, Business, Home Premium (Service Pack 2);
Windows 7 Ultimate,
Professional, Home Premium;
Windows 8 Standard
and Pro edition.
Minimum system requirements are:
Mac OS X 10.6,
Intel-based Mac CPU (Central
Processing Unit), 1 GigaByte (GB) of RAM (Random
Access Memory), 1024x768 or higher resolution
video adapter and display.
Windows XP Professional, Home Edition (Service Pack
3), 700 MegaHertz (MHz) CPU or faster, 256 MegaBytes (MB)
of RAM, 1024x768 or higher resolution video adapter and
display.
A connection to the Internet
is required to display the software tutorial and to
download data for set up, but not to run the tool.
The downloaded file
should be automatically decompressed, generating a "TGCA"
folder.
Failing this, double
click on the file to activate the default decompression
utility of your system.
The
TGCA Folder contains:
"TGCA" (Macintosh) or
"TGCA.exe" (Windows) file
(the runtime application);
"TGCA.fmp12"
(database file);
"FMP Acknowledgments.pdf" file;
"Extensions" folder, containing a
"Dictionaries" folder,
with the
dictionary file for supported languages;
(and an "English" folder with 3 files, for
Windows);
40 ".dll" files (for
Windows).
TGCA
is based on FileMaker Pro 12 (FileMaker Pro, Inc.) database management
software (www.filemaker.com/index.html),
and is
released as a FileMaker Pro 12 template, along with a runtime
application able to run "FileMaker Pro" at the core of the
software.
The runtime is
freely distributed, in compliance with the license of
"FileMaker Pro 12 Advanced" developer package that was
used to create the program.
Standard database commands (Find, Sort, Export
records) are available within each layout of TGCA
(see "GENERAL DEFINITIONS"
and "MENU AND COMMANDS" sections in this
Guide).
Please do not change the names of any files and in
the TGCA folder.
NOTE - Be sure that
your system default format uses
"." (full stop)
as a decimal separator (English
standard).
If this is not the case, you must
change the system setting.
Mac OS X: in
"System Preferences" (from the "Apple" Menu), click on
"International", then on "Formats", then choose as
"Region" a country with the English standard format for
numbers (full stop mark as a decimal separator).
System
restart or user logout is not required to make the
change effective.
Windows: in
"Control Panel" (from the "Start" Menu), click on
"International options" then modify the format of
numbers choosing a country with the English standard
format for numbers (full stop mark as a decimal
separator).
System
restart or user logout is not required to make the
change effective.
SET UP
1. Preparation of expression data
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to Index)
In this section, the user is guided to prepare
and import transcriptome expression data.
1.1)
Export of expression data from TRAM
In order to obtain normalised
gene
expression values, the use of the TRAM (Transcriptome
Mapper) software, able
to generate and analyse transcriptome maps, is
recommended.
TRAM is freely available for download at http://apollo11.isto.unibo.it/software/.
In order to generate
desired transcriptome maps, please follow the TRAM user
guide.
Transcriptome maps already generated through TRAM for
various human tissues are available at http://apollo11.isto.unibo.it/suppl/
(folders: 2014_Caracausi, 2014_Pelleri, 2015_Caracausi,
2016_Caracausi, for details please see the readme.html
file and the corresponding publications).
Example
In order to obtain transcriptome
maps from Acute Megakaryoblastic Leukemia (AMKL) in
children with Down Syndrome (DS) and from normal
megakaryoblasts (MK), go to the web page
http://apollo11.isto.unibo.it/suppl/2014_Pelleri/TRAM_Results/
choose "Mac" or "Win" folder depending on your operating
system and download the
"DS AMKL vs. MK 500 Kb.zip" file.
After the downloaded file decompression, open the TRAM
software by double-clicking on the "TRAM.app" icon and go
to the "Values_A_B" software table by clicking on
the button.
In order to find genes with available expression values
for both conditions, click on the button of the software status
toolbar, type the "=" character in the "Ratio of
Expressions A/B" field , click
on "Omit" and then on "Perform Find" buttons:
Export genes and the corresponding expression values ("A"
represents DS AMKL and "B" represents MK) for retrieved
records choosing "Export Records..." from the "File"
menu:
Choose the type "Tab-Separate Text" and name the file as
preferred.
In the appearing window, select the "Gene_name",
"Expression_A" and "Expression_B" fields from the left square
and click on the "Move" button obtaining the situation
represented in the following figure and lastly click on
the "Export" button.
Alternatively, it is possible to manually create an
expression data file with three columns separated by the
tabulator keyboard button (TAB, ACII19): Gene symbol,
Expression values for "A" condition and Expression values
for "B" condition.
[Column Headers are not required]
[Gene_symbol] [Expression_A]
[Expression_B]
A1BG
21.9222869109593569
41.8496039841932306
A1CF
66.1588030182621625
14.2481863184877172
A2M
30.0147695965851414
13.8910524391818214
1.2) Import of the expression
data in TGCA
Open the TGCA database
and automatically import the expression data by clicking on
the green button "1. Import expression data" and choosing the file created
in the previous step.
2. Preparation of sequences
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In this section, the user is guided through the preparation
and importation of mRNA sequence data.
2.1) Export of sequences
from 5'_ORF_Extender
In
order to obtain mRNA sequences, the use of the
5'_ORF_Extender software,
able to parse RefSeq database, is recommended.
5'_ORF_Extender is freely available for download at http://apollo11.isto.unibo.it/software/.
In order to generate
updated data, please follow the 5'_ORF_Extender user guide.
Human Refseq database data already parsed through 5'_ORF_Extender are available at http://apollo11.isto.unibo.it/software/5'_ORF_Extender/5'_ORF_Extender_2.0/human/
downloading the
filled version depending
on your operating system.
After the downloaded file decompression, open the
5'_ORF_Extender software by double-clicking on the
"5'_ORF_Extender.app" icon and go to the RefSeq_mRNA table
by clicking on the
button.
In order to export only the longest isoform for each gene,
click on the button of
the software status toolbar, select the "SYMBOLUM" and "bp" fields from the left square
and click on the "Move" button (select also Descending
order for the "bp" field) obtaining the situation
represented in the following figure and lastly click on
the "Sort" button.
Export sequences for the longest isoforms for each gene
for sorted records choosing "Export Records..."
from the "File" menu:
Choose the type "Tab-Separate Text" and name the file as
preferred.
In the appearing window, in the "Group by" square check
both "SYMBOLUM" and "bp". Then select the "SYMBOLUM",
"NM_ACCESSION", "Seq_Line" and "bp" fields from the left square
and click on the "Move" button obtaining the situation
represented in the following figure and lastly click on
the "Export" button.
Alternatively, it is possible to manually create a data sequence file with four columns
separated by the tabulator keyboard button (TAB, ACII19):
Gene symbol, NM_Accession, Sequence and length.
[Column Headers are not required]
[Gene symbol] [NM_Accession] [Sequence]
[Length]
A1BG
NM_130786
gggcctcattgct... 1766
A1CF
NM_138933 tttgatatgacga...
9364
A2M
NM_000014
gcacacagagcag... 4678
...
...
2.2)
Importation
of sequences in TGCA
Open the TGCA
database and automatically import mRNA sequences by
clicking on the green button "Import sequences" and choosing the file
created in the previous step.
3.
Description of TGCA Table
(Back to Index)
In
this section, TGCA fields are described.
FIELD
DESCRIPTION
"Gene_Symbol":
the gene symbol;
"NM_Accession":
the mRNA RefSeq
accession number;
"mRNA_sequence":
the mRNA
sequence;
"mRNA_GC_%":
calculate the percentage of GC for each mRNA
sequence
referred to its length; the mean and the standard deviation
in all the records are
shown in the "GC_%_Mean" and
"GC_%_SD";
"GC_count":
calculate the
absolute number of GC for each mRNA sequence;
the sum of this value for all
the records is available at
the bottom of the window in
the yellow section;
"Expression_A":
the
imported expression value for each mRNA in the
condition
"A";
"Expression_B":
the imported expression value for each mRNA in the
condition
"B";
"GC_Count_A":
calculate the multiplication of each GC count for
the
expression value in the condition
"A" for the corresponding
mRNA; the sum of this
value for all the records is available
at the bottom of the window
in the yellow section;
"GC_Count_B":
calculate the multiplication of each GC
count for the
expression value in the condition "B"
for the corresponding
mRNA;
the sum of this
value for all the records is available
at the bottom of the window
in the yellow section;
"mRNA_Length_bp":
the mRNA length in base pairs (bp); the
sum of this value for
all the records is available at
the bottom of the window in
the yellow section;
"mRNA_bp_x_Exp_A":
calculate the
multiplication of each mRNA length for its
expression value in condition
"A"; the sum of
this value for
all the records is
available at the bottom of the window in
the yellow section;
"mRNA_bp_x_Exp_B":
calculate the
multiplication of each mRNA length for its
expression value in condition "B"; the sum of this
value for
all the
records is available at the
bottom of the window in
the
yellow section;
"Transcriptomic_GC_A_%":
calculate
the percentage of GC
in the "B" transcriptome,
which is
the percentage of the "GC_Count_A"
sum in all the
mRNA sequences multiplied
by their expression values in the
condition "B" (sum of the
"mRNA_bp_x_Exp_A"
values for all
records);
"Transcriptomic_GC_A_%":
calculate
the percentage of GC
in the "B" transcriptome,
which is
the percentage of the "GC_Count_B"
sum in all the
mRNA sequences multiplied
by their expression values in the
condition "B" (sum of the
"mRNA_bp_x_Exp_B"
values for all
records).
4. GENERAL DEFINITIONS
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4.1
File
A set of database tables.
4.2 Table
A set of records referring to
the same subject type (e.g., the Gene_Summary
table).
One set of
fields which represents one entry (i.e. containing all requested
data for a subject, e.g. a gene probe).
The record browser is a
small book icon at the top left of the window.
You may also browse the records faster using the cursor to the
right of the small book icon.
4.4. Field
The database unit
containing a specific data type (e.g., 'Gene_ID').
4.5.
Layout
A particular graphical
organization of the field of a table.
A table can be visualized
in more than one layout.
A layout may display
fields from a table or its related fields from other tables.
A file may show data within
different layouts.
Visualization of a field
is independent from the storage of the contained data.
Browsing among the layouts
can be done by clicking on the 'Layout:'
pop-up Menu in the upper left corner.
You may browse the
database by clicking on the small book pages at the top left
of the window, by using the cursor to the right of the small book
icon, or by
entering a record number
and clicking on the "Return" key.
The
following information is constantly displayed on the Status
toolbar (if not, select "Status
Toolbar" from the "View" Menu):
Records: total number of Records in the table.
Found: total number of the subset of Records
currently selected. Clicking
on the green circular button will retrieve the complementary
subset of currently omitted records.
Sorted: sorting status of the Records
(Sorted/Unsorted).
The FileMaker
Pro-based database may be used basically in these "modes":
'Browse', 'Find',
and 'Preview'.
Switching between
different modes can be done from the 'View' Menu or from the pop-up Menu bar at
the bottom left of the window.
4.6
Browse Mode
One way to use the
database.
It allows entry, viewing, browsing,
sorting, and manipulation of data.
It may be selected from
the 'View' menu or
the pop-up mode Menu bar at the bottom left of the
window.
In the 'Browse' mode, the
record sets can be browsed by clicking on the small book icon (with the arrows
to move 'back' and 'forward') in the upper left corner.
Browsing among the tables can
be done by clicking on the 'Layout:' pop-up Menu in the upper
left corner.
An
alternative mode for using the database.
It allows you to search
for specific content in the database fields, using any different
combination of criteria
(see the 'Find mode'
section below for more details).
It may be selected from
the 'View' menu,
the mode pop-up Menu bar at the bottom left of the
window or
the button.
The user can fill in a blank
form allowing to searches in specific fields.
In the "Find" mode, the small
book icon in the upper left corner represents different
"requests" that are made for searching the database.
In FileMaker Pro 'Find'
mode, the "AND" - "OR" - "NOT" operators may be implemented in this way:
"AND" by filling criteria
in different fields
located in the same "Request",
"OR" by generating additional
requests
(from the
"Requests" Menu) in the same query,
"NOT" by generating
additional requests
(from
the "Requests" Menu) and clicking on the "Omit"
button (located on the top bar
in the window).
The 'Operators' pop-up Menu
appears by clicking on a field while pressing the 'ctrl' key, allowing the
query of:
exact matches, duplicate
values, ranges, wild cards and more.
Click on the 'Perform Find'
button at the top of the window to start the query.
The result of the search is the subset of the entries matching
the set search criteria.
Tips:
- we recommend using
the "=" symbol followed
by the gene symbol; for example using the search
"=FAS" in the Gene_Symbol
field will retrieve the gene FAS and the
antisense FAS-AS1;
- in order to search for records with an empty field,
use the "=" symbol not followed by anything in the
desired field.
4.8
Preview Mode
An alternative way of
using the database.
It visualizes a print
preview of the found records.
It may be selected from
the "View" menu
or the pop-up Menu bar at the bottom left of the
window.
In the "Preview" mode, the
user can obtain a print preview of the data in the current table.
Browsing among the
tables can be done by clicking on the 'Layout:' pop-up
Menu in the upper left corner.
About
FileMaker Pro Runtime...
Information about
FileMaker Pro Runtime at the core of the software.
Preferences...
Standard preferences
panel; cache memory size can be set at up to 256 Mb.
Hide
TGCA
Hides all TGCA windows.
Quit TGCA
Closes the programme.
File
Options...
It is only possible to
set the "Spelling" options.
Change
Password...
There is no default
password set.
Page
Setup...
Standard page set up
command.
Print...
Standard print command.
The appearance will
match the layout
currently displayed on the screen.
Import
Records
This is the general
"Import" function of FileMaker Pro.
Export
Records...
Export command for the found records set in a
given table.
Records are exported in
their current sorting mode.
User can select fields
to be exported, their relative order,
and the separation
character.
Save/Send
Records As...
Saves records to a
specified Excel worksheet.
Send
Sends an intranet or
internet email message (with or without a file attachment)
to one or more recipients.
Email can be sent
through an email application or via SMTP (Simple Mail
Transfer Protocol, a set of criteria for sending and
receiving email).
Save a
Copy as...
Save a copy of the database, compacted,
compressed, as
a clone (database structure with no record present) or as a
self-contained copy.
5.3
'Edit' Menu
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Undo
Standard "Undo" command.
Redo
Standard "Redo" command.
Cut
Standard "Cut" text
command.
Copy
Standard "Copy" text
command.
Paste
Standard "Paste" command.
Paste
Text Only
Standard "Paste" text
command.
Clear
Deletes the contents of
the specified field in the current record.
Select
all
Selection of all text
present within a selected field
(to select a field, click
on the field).
Find/Replace
Utility for
searching/replacing text strings within fields.
Note: Use 'Find' mode
(from the "View" Menu) for full search and selection of a record set.
Spelling
Function for checking
spelling of text strings within fields.
Export
Field Contents...
Function for exporting the
contents of the selected field to a file.
5.4
'View' Menu
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Browse
Mode
Switch to the 'Browse
Mode' (see "General Definitions" above).
Find
Mode
Switch to the 'Find
Mode' (see "General Definitions" above).
Preview
Mode
Switch to the 'Preview
Mode' (see "General Definitions" above).
Go to
layout
A possible way to switch
between different layouts.
View
as Form
A possible way to individually display the current record
of a found set
of records.
View
as List
A possible way to
display all the records of a found set in the form of a
list.
View
as Table
A possible way to
display all the records of a found set in the form of a spreadsheet-like table.
Status
Toolbar
To switch on/off the
"Status Toolbar": the toolbar located at the top of the
programme window.
Customize
Status Toolbar
To customize the "Status
Toolbar" buttons.
Formatting
bar
To switch on/off the
"Formatting Toolbar".
Ruler
To switch on/off the
text ruler of the application.
Zoom
in
Used to increase layout
dimensions.
Zoom
out
Used to decrease layout
dimensions.
5.5
'Records' Menu
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New
Record
Creates a new empty
record in the database.
The new Record will be
the latest in the current record set.
Duplicate
Record
Duplicates the current
record in the database.
The new Record will be
the latest in the current record set.
Delete
All Records
Deletes all the records
in the database.
Delete
Found Records...
Deletes all currently
found records in the database.
Go to
Record
Moves to the selected
record by number, previous or next.
Refresh
Window
Updates the entire
contents of the database window, including any related
records.
Show All
Records
Shows all the records in
the database.
Show
Omitted Only
Shows all the records in
the database not included in the current found set.
Omit
Record
Removes the selected
record out of the current found set, without deleting it.
Omit
Multiple...
Removes more than one
record, selected by numbers, out of the current found set, without deleting
them.
Modify
Last Find
Returns to the last
performed search in order to edit it.
Saved
Finds
Saves a set of search
criteria.
Sort
Records...
Sorts the current record
set according to desired criteria.
In the Sort Records dialog box, from the left square choose
the desired table fields available for sorting in the order you want them sorted and
click on the "Move" button. Clicking on the "Clear" button
will delete previously chosen fields. By default, the
records remain sorted until you perform a find or sort
records by different criteria.
This
function is available also clicking on the button on the Status
Toolbar.
Unsort
Displays the current
record set according to the order of creation of each
record.
Replace
Field Contents...
Replaces the value of a
field in all sets of records found with the value specified in the
current record, or by calculation.
Relookup
Field Contents
This
command updates the value of a field by reading the matched
value in a related table (the relationship has been
established during database development using a 'key'
field).
Revert
Record...
Restores the value of a
field, discarding any change, before clicking out of that
field.
5.6 'Scripts'
Menu
(Back to Index)
Tutorial
The
page with the TGCA tutorial.
Import_Expression_Data
Please see section 1.2.
Import_Sequences
Please see section 2.2.
5.7
'Help' Menu
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Search
Search a 'Help' system for the general commands.
TROUBLESHOOTING
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Sometimes,
power failure, hardware problems, or other factors can
damage a
FileMaker Pro database file.
When the runtime
application discovers a damaged file, a dialog box appears, prompting the user to
contact the creator.
Even if the dialogue box
does not appear, files can exhibit erratic behaviour.
If you have FileMaker
Pro or FileMaker
Pro Advanced
installed you can recover them using the 'Recover'
command.
Otherwise, to recover a damaged file:
- On Mac OS X machines,
press Command + Option (cmd-alt) while double-clicking the runtime application icon. Hold the keys down until
you see the 'Open Damaged File' dialogue box.
- On Windows machines, press
Ctrl+Shift while double-clicking the runtime application
icon. Hold the keys down until you see the Open Damaged File
dialogue box.
During the recovery
process, the runtime application:
1. Creates a new file;
2. Renames any damaged files
by adding “Old” to the end of the
file name;
3.
Gives the repaired file the original name.