Lasyf|g Manua|

Vers|on 2.1 (16 Apr|| 2012)

M|tche|| I. Su|||van, N|co|a k. etty and Scott A. 8eatson. (2011)
Lasyf|g: a genome compar|son v|sua||ser. 8|o|nformat|cs, 27 (7):
1009-1010
!""#$%%&'()*+,-(./01&*.0,&-2&"%

2
Lasyf|g

Easyfig is a Python application foi cieating lineai compaiison figuies of multiple
genomic loci with an easy-to-use giaphical usei inteiface (u0I). BLAST compaiisons
between multiple genomic iegions, ianging fiom single genes to whole piokaiyote
chiomosomes, can be geneiateu, visualiseu anu inteiactively colouieu, enabling a
iapiu tiansition between analysis anu the piepaiation of publication quality figuies.

42("'55'"+.2

Microsoft Windows: Download the latest version oI EasyIig (currently
EasyIigwin2.1.zip). Extract contents to the desired location. From within the
downloaded Iolder run EasyIig by double-clicking on the EasyIigwin2.x.exe
executable Iile; ensure that the Iolder has been Iully extracted and that
EasyIigwin2.x.exe is being run Irom within the Iolder. On Windows Vista/7,
the EasyIig Iolder may appear as an application rather than a Iolder: simply
double-click the application to launch EasyIig (known to work on Windows
Vista/7 and on PCs younger than 4 years old running Windows XP).

Mac OS X: Download the latest version oI EasyIig (currently
EasyIigmac2.1.tar.gz) and double click on the download to extract contents.
Double-click on the EasyIig2.x application to run EasyIig (known to work on
Mac OS X versions 10.5 and 10.6).

Fedora/Ubuntu/Linux 64 bit: Download the latest version oI EasyIig (currently
EasyIiglinux2.1.tar.gz). Extract contents to the desired location. From within the
downloaded Iolder run EasyIig by double-clicking on the EasyIiglinux2.x
binary Iile; ensure that the Iolder has been Iully extracted and that
EasyIiglinux2.x is being run Irom within the Iolder.

There are some known issues running compiled versions of Python
applications on Windows/Mac OS X. If Easyfig does not run using the above
methods we recommend using the Linux/Unix/Windows cmd version.

Linux/Unix/Windows cmd: EasyIig2.x.py is executable Irom a terminal
window in any Unix machine (including Mac OS X). Download the latest version
oI EasyIig (currently EasyIig2.1.py) and save to desired location. Open a
terminal window, navigate to the directory where you saved EasyIig and type
'python EasyIig2.1.py' on the command line; this will launch the GUI.
Alternatively, EasyIig2.x.py can be run on Windows or Mac OS X by right-
clicking and choosing open with Python or Python Launcher. Using this version oI
S
EasyIig requires Python 2.5 or later to be installed. This is available at
http://python.org. The graphical user interIace library used by EasyIig (Tkinter) is
not included with some distributions oI Linux and Mac OS X. We recommend
reinstalling Python 2.7 Irom the Python website as Tkinter is included in the
standard library.

Command-line version: EasyIigCL2.x.py is identical to EasyIig2.x.py except
that it does not launch the EasyIig GUI. BLAST and Figure-drawing parameters
are set using command-line arguments. Running EasyIigCL2.x.py with no
arguments will print to screen the available options Ior EasyIig.

6&""+2, /# 7'()*+, ". '/".8'"+1'55) ,&2&0'"& 9:;6< *+5&($

EasyIig can run using pre-computed BLAST comparison Iiles, but it is also
capable oI generating BLAST comparisons iI BLAST executables have been
installed.

EasyIig can automatically install BLAST executables. From the menu bar click
'Blast' and choose 'Download Blast Automatically'. This may take a while on
slower connections as the download is large (~ 50 MB). II BLAST installs
correctly you will be presented with a message saying 'BLAST installed', you
will now be able to automatically generate BLAST comparisons using the
generate buttons.

II an FTP connection cannot be created or the correct binaries Ior your system
cannot be Iound you will be presented with a message asking you to download
BLAST manually. To do so click 'Blast' in the menu bar and choose 'Download
Blast Manually'. This will bring up a message box recommending a version oI
BLAST to download, clicking 'ok' will open the download page in your browser.
4
0iganisation of this manual:

='0" >$ </".0+'5(

We stiongly iecommenu that all new useis follow the examples below to familiaiise
themselves with Easyfig.

='0" ?$ @&'"/0&( .* 7'()*+,

All options anu scieens available in Easyfig aie uesciibeu in uetail.

='0" A$ B.88'2CD5+2& .#"+.2(

0ptions available using the commanu-line (non-giaphical usei inteiface) veision of
Easyfig



















S
art 1: 1utor|a|s

Please ensure that Iiles are loaded in the order speciIied below. Words/characters
in parentheses e.g. (open) reIer to button labels

Example files (Easyfig_example1_files.zip anu Easyfig_example2_files.zip) can be
uownloaueu fiom the SouiceFoige Easyfig uownloau page at
http:souicefoige.netpiojectseasyfigfiles


Example 1. Simple genome region comparison

In this example we will compare two ~50 Kb prophage regions Irom diIIerent
!"#$%&'##" genomes. The Iiles needed Ior this example are in
EasyIigexample1Iiles. (available to download Irom
http://sourceIorge.net/projects/easyIig/Iiles/)

A: Comparison of two genomic regions.

1. Launch EasyIig.

LOAD ANNOTATION FILES:
2. Click the Add Ieature Iile button immediately underneath the Annotation files
box to navigate to the Easyfig_example1_files Iolder

3. Select the Newport_Gifsy.gbk Iile and click ( open )

4. Click the Add Ieature Iile button again

5. Select the LT2_Gifsy.gbk Iile in the Easyfig_example1_files Iolder and click
( open )

LOAD BLAST COMPARISON FILE:
6. Click the Add blast Iile ( ) immediately underneath Blast files box;
navigate to the Easyfig_example1_files Iolder and select:
Newport_Gifsy1_v_LT2_Gifsy1.blastn.out

SPECIFY OUTPUT FILE
7. Click ( Save as ), enter example1.bmp in the box and click ( Save )

6
CREATE FIGURE
8. Click ( Create Figure ). EasyIig progress will be displayed in the yellow dialog
box.

9. Open the Iolder where you saved the EasyIig image and open it with your
Iavourite image-viewer.



B: Invert the LT2 region to obtain a clearer comparison

1. Click Image on the menu bar and select Subregion

2. In the popup window, double-Click on the second Iile, select the Reverse
radio button and click ( Save & Close )

3. Close the Subregion window using (close)

4. CREATE FIGURE as described in part 1A.



C: Colour the coding-sequence (CDS) features

1. Click Image on the menu bar and select Annotation

2. Unclick the gene radio button under the Include following features section
oI the Annotation Window

3. Click the CDS radio button
7

4. (Optional) Add a scale by entering 5000 in the Length of scale legend (in base
pairs) box Iound in the Iigure window accessible Irom Image dropdown menu.

5. CREATE FIGURE as described in part 1A.



Colours can be added to any Ieature Irom within EasyIig using a pop-up colour
palette, or by adding a /colour Ieature qualiIier to the annotation Iile (as has been
done Ior these examples). The /colour qualiIiers can be added using an annotation
tool such as Artemis www.sanger.ac.uk/resources/soItware/artemis/, or by editing
the annotation Iile in a text editor. Both Artemis colour codes (0-18) and standard
RGB colour codes (/colour255 0 0) are interpreted by EasyIig.

In the example Iiles loaded in parts A and B, the Artemis colour codes have been
added to the CDS Ieatures e.g.

CDS 4038..4265
/locus_tag="STM2585A"
/note="similar to pagK"
/codon_start=1
/transl_table=11
/product="PagK-like protein"
/protein_id="NP_461521.1"
/db_xref="GI:16765906"
/db_xref="GeneID:1254108"
/colour=10

By deIault, gene Ieatures are shown, which is why all the Ieatures are the same
colour in exercise A and B.


8
D: Zoom in on a ~15kb subregion at one end of the sequences

1. Access the subregion window Irom the Image dropdown menu; click on Iile 01
and then enter 32599 and 50099 in the Min and Max Range boxes located
directly under the list oI annotation Iiles

2. Click (change cutoIIs)

3. Click on Iile 02 and then enter 1 and 15000 in the Min and Max Range boxes
and click (change cutoIIs)
IMPORTANT: remember that the second Annotation Iile (LT2_Gifsy.gbk)
has been inverted, this has been taken into account when enteiing the subiegion
iange

4. Close the subiegion winuow with (close)

5. Click on ( Generate blastn Files ) to generate the BLAST comparison Iiles Ior
these subregions (a pop-up box will ask you where you want to save the BLAST
Iiles deIault is usually in the Easyfig_example1_files Iolder)

4. CREATE FIGURE as described in part 1A.
In the new image, the scale is still 5000 bp, but the image is zoomed in on the
variable region. The minimum BLAST identity value is shown in the yellow
dialog box aIter each Iigure is drawn this can be used to calibrate the BLAST
identity scale shown on the right (i.e. in this case the matches range Irom 100
(darkest) to 85 (lightest)).



As described in the manual there are many ways to customise the image Iurther
e.g. the Iollowing list shows a Iew oI the options available:
- Feature rendering type Irom arrows to boxes or pointers
- Colours oI any oI the Ieatures
- Thickness oI any lines
- Height oI BLAST matches
- Height oI Ieatures
9
- Width oI Iigure
- Type oI image Iile (bmp is deIault, but svg |scalable vector graphics| Iiles can be
produced by changing the Iile type)
- Add a Ieature legend or label the Ieatures


1u
Example 2. Whole genome comparison

Example Iiles are contained in the EasyIigexample2Iiles Iolder (available to
download Irom http://sourceIorge.net/projects/easyIig/Iiles/)

In this example we are creating a comparison Iigure oI three )*+,'-.+,." +%#.
genomes, O157:H7 EDL933, O157H7 Sakai and K12 MG1655. We want to
illustrate the phage regions, but as these are not labeled uniquely in the original
GenBank Iiles we have added them manually using phage as a feature label. For
example in the K12 Iile we add 12 lines, one Ior each phage:


FEATURES Location/Qualifiers
source 1..4639675
/organism="Escherichia coli K12"
/mol_type="genomic DNA"
/strain="K-12"
/sub_strain="MG1655"
/db_xref="taxon:83333"
phage 262182..296489
phage 1195443..1210646
etc

The phage Ieatures have already been added to the Iiles provided:
EcSakai.withPhage.gbk, EcEDL933.withPhage.gbk, EcK12.withPhage.gbk.

A. Comparison of three "# $%&' genomes

1. Launch EasyIig. (II continuing Irom Example 1, quit EasyIig beIore starting)

2. LOAD ANNOTATION FILES as described in part 1A, in this order:
EcSakai.withPhage.gbk
EcEDL933.withPhage.gbk
EcK12.withPhage.gbk

3. LOAD BLAST COMPARISON FILES as described in part 1A, in this order:
Sakai_EDL933.blastn.out
EDL933_K12.blastn.out

4. SPECIFY OUTPUT FILE as described in part 1A


11
5. CREATE FIGURE as described in part 1A.


IMPORTANT: By deIault, EasyIig does not display Ieatures or BLAST matches
smaller than 4 pixels. This feature can be turned off and on using the Filter small
blast hits/annotations button Iound in the Blast option Irom the Image dropdown
menu. II this Iilter is turned oII with a whole genome sequence comparison the
thousands oI small, insigniIicant BLAST matches and Ieatures will swamp the
image and greatly prolong rendering times. It is recommended that you turn this
Iilter oII and use the BLAST options and custom Ieatures (as described in Part B)
to Iilter the small BLAST matches and Ieatures in whole genome comparisons.

B. Display custom features on a whole genome comparison

1. Access the annotation window by choosing annotation Irom the Image
dropdown menu and uncheck the gene radio button under Include following
features

2. Enter the word phage into the open custom feature box under Include
following features and click the radio button on the right of the box

3. Click on Colour button ( ) to the right of the custom feature box and change
to your Iavourite colour

4. Use the pull-down menu to the Iar right oI the custom Ieature box and choose
rect for rectangle

5. Close the annotation window using the (close) button

6. Access the blast window by choosing Blast Irom the Image dropdown menu

7. Enter 10000 under Min. length
12

8. Unclick the Filter small blast hits/annotations button

9. CREATE FIGURE as described in part 1A.



C. Change the BLAST identity gradient and show a graph

1. In the Blast window Irom the Image dropdown menu, click the colour buttons (
) and choose colours Ior minimum and maximum colours Ior normal and
inverted BLAST matches:
normal-minimum: aqua
normal-maximum: blue
inverted-minimum: orange
inverted-maximum: red
(the exact colours you choose is not important, as long as there is a contrast
between the normal and inverted BLAST matches)

2. Unclick the Outline blast hits in black

3. Access the Graph window by choosing graph Irom the Image dropdown menu

4. Choose GC content from the pull-down Graph: menu


1S
5. CREATE FIGURE as described in part 1A.


14
art 2: Ieatures of Lasyf|g
Ma|n Screen



Above ls Lhe wlndow avallable upon lmmedlaLely openlng Lasyflg. verslons may vary
sllghLly as Lhe graphlcal user lnLerface uses a sLyle naLlve Lo Lhe operaLlng sysLem ln use.

Annotat|on I||es: A llsL of Cen8ank, LM8L, lAS1A or mulLllAS1A flles you wlsh Lo creaLe
a flgure from.

8|ast I||es: A llsL of 8LAS1 flles for drawlng Lhe comparlson beLween Lwo annoLaLlon
flles. 1he annoLaLlon flle above musL be Lhe query, and Lhe annoLaLlon flle below musL
be Lhe reference. valld lnpuL flles are legacy blasL and blasL+ Lab dellmlLed ouLpuL
wlLhouL headers and crunch flles. llles musL be made uslng 8LAS1n or L8LAS1x. 1hese
are made uslng Lhe -ouLfmL 6 Lag ln blasL+ or m 8 Lag ln legacy blasL.

Generate b|astn]tb|astx I||es: Wlll generaLe 8LAS1n or L8LAS1x comparlsons beLween
loaded annoLaLlon flles. 8equlres 8LAS1 Lo be lnsLalled, see above for lnsLallaLlon
lnsLrucLlons.
1S
lMC81An1: L8LAS1x ls very memory expenslve and lL may noL be posslble Lo perform
on large genomes. We recommend uslng lL Lo compare smaller reglons only (< 300 kb).

Save As: 1hls changes Lhe locaLlon of where your flgure wlll be creaLed. Changlng Lhls
afLer your flgure has been creaLed wlll noL save lL agaln under Lhe new name.

I||e type: Lasyflg flgures may be saved as 8lLmap (bmp) or Scalable vecLor graphlcs
(svg). SvC flles requlre an lmage vlewer such as phoLoshop or ClM, buL are much
beLLer quallLy Lhan bmp flles and easler Lo manlpulaLe. We recommend lnkscape, a free
open source lmage edlLor for vlewlng and manlpulaLlng SvC flles.

Create I|gure: CreaLes Lhe flgure aL Lhe locaLlon speclfled ln Save As. Wlll overwrlLe
prevlous flgures wlLhouL warnlng

I||e: lor savlng and loadlng seLLlngs or preferences

Image: Access wlndows for modlfylng Lhe flgure belng creaLed

8|ast: Access opLlons Lo manually or auLomaLlcally download 8LAS1

ne|p: lnformaLlon abouL uslng and referenclng Lasyflg

16
Image I|gure



W|dth of f|gure: 1he wldLh of Lhe lmage generaLed ln plxels.

1h|ckness of genome ||ne: 1hlckness ln plxels of Lhe llne represenLlng Lhe full sequence.

ne|ght of genes |n f|gure: 1he helghL of Lhe annoLaLlon parLs of Lhe flgure.

ne|ght of b|ast h|ts |n f|gure: 1he helghL of Lhe reglons showlng blasL maLches. 1he LoLal
helghL of Lhe flgure generaLed wlll be (no of annoLaLlon flles x helghL of genes ln flgure)
+ (no of blasL flles x helghL of blasL hlLs ln flgure).

A||gnment of genomes: LefL, rlghL, cenLre, besL blasL. Cenomes can be allgned on Lhe
lefL, Lhe rlghL or cenLered ln Lhe mlddle of Lhe flgure. AlLernaLlvely Lhey can be allgned
so LhaL each genome ls perpendlcular Lo Lhe ad[acenL genomes aL Lhe slLe of Lhelr besL
blasL hlL.

Draw b|ast |dent|ty |egend: uraws a colour gradlenL legend for vlsuallslng Lhe ldenLlLy of
your blasL hlLs.

Sca|e |egend: uraws a P brackeL of Lhe slze speclfled ln base palrs for help vlsuallslng Lhe
length of your genomes. The default 0 will mean there is no scale legend drawn.

Ieature Legend: CreaLes legend of all Lhe feaLures used ln Lhe lmage.

Get feature names from: CeLs feaLure names from Lhls descrlpLor ln Lhe genbank/embl
flle
17
Image Annotat|on


Ieature |abe|s: Label feaLures (lnformaLlon on feaLure labels can be found aL Lhe end of Lhls
secLlon)

Get feature name from: CeLs feaLure names from Lhls descrlpLor ln genbank/embl flle

1h|ckness of exon ||ne: 1hlckness of Lhe llne ln plxels [olnlng exons, or segmenLs of a pseudo
gene.

1h|ckness of gene out||ne: 1hlckness of Lhe black ouLllne around Lhe genes.

Inc|ude fo||ow|ng features: 1lck checkbox for Lasyflg Lo draw any feaLure of Lhe Lype
represenLed. 1he lasL box allows Lhe user Lo lnpuL a feaLure Lype. 1hls ls case senslLlve.

Co|our: wlll be Lhe defaulL colour used lf no colour annoLaLlon can be found ln Lhe feaLure flle.
Colour Lags are of Lype /colour= And can elLher be 3 spaced lnLegers ln 8C8 form or colour
coded uslng Lhe ArLemls colour codes (lnLegers ranglng from 1 Lo 18).

1ype: Pow Lo lllusLraLe each feaLure.


Arrow: ulrecLlon arrows of feaLures.


kect: 8ecLangle of Lhe lengLh of Lhe feaLure.


Irame: ulrecLlonal arrows ln frame.


o|nter: olnLers aL Lhe sLarL of each feaLure.
18
Image 8|ast



M|n. |ength: Mlnlmum lengLh of blasL hlLs Lo be drawn.

Max. e Va|ue: Maxlmum expecL value of blasL hlLs Lo be drawn.

M|n |dent|ty Va|ue: Mlnlmum ldenLlLy value of blasL hlLs Lo be drawn.

Choose m|n|mum]max|mum b|ast co|our: 8LAS1 hlLs are coloured on a gradlenL wlLh
mlnlmum 8LAS1 colour belng Lhe colour of 8LAS1 hlLs wlLh Lhe mlnlmum ldenLlLy
reporLed and maxlmum 8LAS1 colour belng Lhe colour of 8LAS1 hlLs wlLh 100 ldenLlLy.
normal ls Lhe colour for forward-forward and reverse-reverse maLches. lnverLed ls Lhe
colour for forward-reverse blasL maLches.

Cut||ne b|ast h|ts |n b|ack: 8y defaulL, elLher slde of Lhe blasL hlL ls ouLllned by a solld
black llne. 1hls helps dlsLlngulsh beLween blasL hlLs buL may obfuscaLe ldenLlLy for
smaller blasL hlLs. uncheck Lo Lurn off.

I||ter sma|| b|ast h|ts]annotat|ons: lllLer 8LAS1 hlLs or feaLures under 4 plxels wlde.
8educes nolse for lnlLlal vlew of comparlson. lor example, wlLhouL Lhls opLlon, whole
genome comparlsons are unreadable due Lo all Lhe low slgnlflcance maLches, and
Lhousands of feaLures. Cnce famlllar wlLh Lhe lmage, lL ls recommended LhaL users Lurn
Lhls off and use 8LAS1 opLlons and cusLom annoLaLlons Lo reduce Lhe complexlLy of Lhe
lmage (as descrlbed above ln example 2).
19
Image Graph





Graph: SelecL Lype of graph.
GC content: ercenLage of guanlnes and cyLoslnes ln each wlndow, glven by (C +
C) / (C + C + A + 1) where C, C, A and 1 are Lhe counL of each nucleoLlde.
GC skew: 8aLlo of guanlnes Lo cyLoslnes ln each wlndow, glven by (C C) / (C + C).
Coverage: lf provlded wlLh an ACL flle ln Lhe lnpuL flle fleld, Lasyflg wlll calculaLe
Lhe coverage across each conLlg, and ploL lL agalnsL Lhe flrsL ldenLlcal sequence
found ln Lhe feaLure flle. Can only be used wlLh Lhe Lop genome.
Custom: loLs a cusLom graph. lnpuL ls a plaln LexL flle wlLh a slngle column of
numbers, one llne per daLa polnL.
lf more daLapolnLs are provlded Lhan Lhe wldLh of Lhe lmage, daLapolnLs correlaLlng Lo
each plxel wlll be averaged.

Mu|t|p|e graphs: lf unchecked only Lhe Lop sequence flle wlll be graphed. lf checked all
sequence flles wlll be graphed. 1o ploL mulLlple cusLom graphs, Lhe lnpuL flle should
have numbers ln columns separaLed by Labs for each sequence flle Lo be ploLLed.

Input f||e: Clve Lhe fllename of Lhe user creaLed cusLom flle, or an ACL flle.
2u

Step: CeneraLe a daLa polnL every x base palrs.

W|ndow s|ze: AmounL of bases ad[acenL Lo each sLep Lo calculaLe CC conLenL/skew. 1hls
ls Lhe amounL of bases ln LoLal Lo be counLed l.e. 1000 counLs 300 bases Lo each slde of
Lhe sLep poslLlon.

Max|mum : maxlmum value of Lhe Y axis. If set to Auto thls wlll be seL Lo Lhe
maxlmum of Lhe ? axls wlll be seL as Lhe maxlmum ? value.

Log Sca|e (|og 10): Make Lhe ? axls log10 scaled. useful for hlgh coverage daLa.

Graph type: Choose hlsLogram or llne graph.

Ax|s ||ne th|ckness: 1hlckness of Lhe x axls llne ln plxels.

os|t|ve va|ue co|our: Colour of Lhe llne or hlsLogram bars wlLh poslLlve y values or CC
conLenL greaLer Lhan 30.

Negat|ve va|ue co|our: Colour of Lhe llne or hlsLogram bars wlLh negaLlve y values or CC
conLenL lower Lhan 30.

Gap between graph and f|gure: Cap ln plxels beLween Lhe graph and Lhe annoLaLlon
and comparlson drawlngs.

21
Image Subreg|ons



List of subiegions of annotation files to use. Cutoffs may be changeu foi highlighteu
value by enteiing new values into the input boxes below the lists anu clicking
(change cutoffs). Bouble-clicking a file will also biing up a box to change the cutoffs.


22
Legend opt|ons
@&'"/0& 5&,&2C (available fiom ImageFigure)
Lists all genes (genes appeaiing in multiple genomes aie only listeu once) unuei the
figuie in a single oi two columns. Naximum genes listeu is 1uu, maximum length of
gene name is Su chaiacteis. Featuie legenus cannot be useu in conjunction with
featuie labels.



@&'"/0& 5'E&5( (available fiom ImageAnnotation)
Labels genes (labels largest gene if label doesnt fit) on the top and/or bottom
genome. Naximum length of gene name is 1u chaiacteis. Boes not woik in
conjunction with featuie legenu. Cannot label top genome anu incluue giaph.



2S
I||e New I|gure

Removes all files anu iesets piefeiences to uefault
I||e Save Sett|ngs

Saves all settings incluuing piefeiences anu filenames
I||e Load Sett|ngs

Loaus a pieviously saveu configuiation
I||e references




Save preference as: lf no flle exlsLs aL ./.easyflg.pref creaLes one. Saves all lmage
preferences (buL noL flle names)

Load preferences: Loads prevlously saved lmage preferences, does noL change flles
loaded.

kemove: 8emoves a preference

Set as defau|t: references loaded aL sLarL up (easyflg_sLandard orlglnally)




24
art 3: Command-||ne opt|ons

Easyfig_CL_2.1.py Written by: Mitchell Sullivan mjsull@gmail.com
Supervisor: Dr. Scott Beatson & Dr. Nicola Petty, University of Queensland
Version 2.1 20.04.2012

License: GPLv3

Usage: Easyfig_CL_2.1.py [options] GenBank/EMBL/fasta GenBank/EMBL/fasta
GenBank/EMBL/fasta ...

This script should work on 1 to an infinite amount of GenBank/EMBL files (given
enough memory)

Adding 2 integers after the annotation file will crop the annotation file.
Adding a R after the annotation file will reverse compliment it.

WARNING: Will overwrite output file without warning.

***************************************************************
GenBank or EMBL file must have source line, or Sequence.
' source 1..<sequence length>' or 'FT source 1..<sequence length>'

for GenBank / EMBL

***************************************************************

The GenBank file preceding the blast file should always be the query
the GenBank file after the blast file should always be the reference
In it's present state only 'CDS' features will be recorded

Options:
-o <string> Specify output file. <REQUIRED!>
-blastn Generate blastn files automatically. Requires blastall or blast+
in the path, Annotation file must have nucleotide sequence.
[Default]
-tblastx Generate tblastx files automatically. Requires blastall or blast+
in the path, Annotation file must have nucleotide sequence.
-blast_files List of previously generated blast files, ordered. Query must be
annotation file on top, reference annotation file on bottom.
-svg Create Scalable Vector Graphics (svg) file instead of bmp.
-filter Filter small blast hits or annotations (< 4 pixels wide). [F]


GENERAL OPTIONS:
-width <int> width of figure in pixels. [5000]
-ann_height <int> height of annotations in figure (pixels). [50]
-blast_height <int> height of blast hits in figure (pixels). [100]
-f1 <T/F> draw colour gradient figure for blast hits. [F]
-f2 <int> draw scale figure <int> base pairs long. [0]
-uncomp <T/F> Do not compress figure. [F]
-f <string> [r g b] [arrow/rect/pointer/frame]
Draw features of type <string> (case sensitive) in the
color r g b with illustration type arrow, rectangle,
pointer or frame. Default light blue arrows.
EXAMPLE: -f CDS 255 0 0 rect will draw all CDS features
as
a red rectangle.
if none specified easyFig automatically draws CDS
features.
If you want a figure with no features drawn use -f F
2S
-glt <int> Genome line is <int> pixels thick [5]
-exont <int> exon lines joining introns are <int> pixels thick. [1]
-genet <int> outline of features is <int> pixels thick. [1]
-aln <best/left/right/centre> [centre]
Alignment of genomes
best aligns feature file perpendicular to best blast hit.
-legend <single/double/top/bottom/both/None>
Single: Gene names in single column
Double: Gene names in two columns
Top: Top feature file genes labelled above figure
Bottom: Bottom feature file genes labelled below figure
Both: Top and bottom feature files genes labelled above
and below genome.
None: No legend or gene labelling <default>
-leg_name Where to get feature name from [gene]

BLAST OPTIONS:
-e <float> maxmimum e value of blast hits to be drawn. [0.001]
-i <float> minimum identity value of blast hits to be drawn. [0]
-min_length <int> minimum length of blast hits to be drawn. [0]
-blast_col <red/blue> changes blast hits to gradient of red or blue
alternitively <int1 int2 int3 int4 int5 int6>
defines color gradient for blast hits
worst blast hit reported will be color int1 int2 int3
where int 1 2 3 is the RGB of color range[0-255]
100% identity blast hits will be color int4 int5 int6
[default 20 20 20 175 175 175] <gray>

-blast_col_inv Colour for inverted blast hits.
-bo <T/F> Black outline of blast hits. [T]

GRAPH OPTIONS:
-G <GCContent/GCSkew/Coverage/Custom [filename]>
Plot GC Content, GC Skew, Coverage or Custom graph.
if Coverage or Custom filename for ace or custom file
needs
to be provided. Details on how to make custom graph files
in manual.
-wind_size <int> Window size for calculating GC content/GC skew. [1000]
-step <int> Step size for calculating GC content/GC skew. [1000]
-line <T/F> Draw graph as a line graph. [T]
-axis_t Thickness of X axis. [1]
-pos_col <int int int> RGB colour of positive values in graph. [Red]
-neg_col <int int int> RGB colour of negative values in graph. [Blue]
-g_height <int> height of graph in pixels. [50]
-gap gap between graph and annotations. [10]
-y_max Maximum y value [Default: max Y calculated.]


EXAMPLES:

Easyfig_CL_1.2.py -filter -o outfile.bmp genbank1.gbk genbank2.gbk genbank3.gbk

Easiest way to generate a simple comparison file between three (or more)
annotation
files. Shows CDS features as red arrows.

Easyfig_CL_1.2.py -o outfile.bmp -e 0.00001 -f gene frame 0 0 255 -G GCContent
ann1.embl ann2.gbk ann3.gbk ann4.embl

Generate a blastn comparison between 4 annotation files, Display genes as blue
arrows in frame. Only report blast hits under 0.00001 expect value.
Display the GC content of each file as a graph.
26

Easyfig_CL_1.2.py -tblastx -o outfile.svg -svg ann1.embl 1 10000 ann2.embl 1
10000 R

Show a tblastx comparison of the first 10000 base pairs of ann1.embl and
ann2.embl
Reverse compliment ann2.embl. Writes as a SVG file.


I|na| comments

Please email the authoi at mjsullgmail.com if piogiam ciashes oi bugs aie founu.
Console piintout anu shoit uesciiption of what was being attempteu is appieciateu.

Feel fiee to email if you have any iueas foi futuie implementations (othei commonly
useu giaphs, foimats to make compatible, othei illustiation options)

Thank you foi using Easyfig,
The Easyfig team