step1.html000664 022642 001631 00000003046 06505776233 013435 0ustar00valYeasties000000 000000 Overview of the features map

Overview of the features map

With the GeneFinder Tutorial database open....

Double click on the Test_Sequence class in the Classes window and then double click the object EM:D13249 in the KeySet window. The features map (fmap or sequence map) appears.

Now click on the GeneFind.. button at the top right of screen. A message box appears; press the Continue button. GeneFinder features appear; these are stop codons, ATG codons, blocks of coding potential, splice donor and acceptor sites (see diagram below)

With the right hand mouse button click on the GeneFind.. button and select [AutoFind gene] from the menu that appears. A message box appears giving the maximum and minimum co-ordinates of the search, and the score for the "temp_gene" which was found; press Continue. The temp_gene is the GeneFinder prediction and is marked on the fmap.

Some GeneFinder features are now marked in green. These are the ATG (AUG) translation initiation codon, splice donor and acceptor sites and an open reading frame corresponding to the terminal exon of this predicted gene. Note that the GeneFinder prediction does not correspond to the gene from the EMBL record.

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map.

Some GeneFinder features are now marked in green. These are the ATG (AUG) translation initiation codon, splice donor and acceptor sites and an open reading frame corresponding to the terminal exon of this predicted gene. Note that the GeneFinder prediction does not correspond to the gene from the EMBL record.

Go to Step 2


Returstep10.html000664 022642 001631 00000006536 06505776233 013524 0ustar00valYeasties000000 000000 The End

The End

The GeneFinder Tutorial database contains 38 Test_Sequences together with regions of homology determined by BlastX, BlastN, TBlastX and PSsearch. If you would like some practice at GeneFinding, have a go at these and compare your results to the table below. User segments of splice branch/acceptor motifs are available in the GeneFinder_Tutorial.user_segments/ directory for each of the sequences.

Of the 32 protein coding genes specified in the EMBL feature tables of the Test_Sequences, GeneFinder correctly predicts 24 (75%), with a single set of GeneFinder parameters (see below). 39 introns from a total of 41 (95%) are correctly predicted, with only 5 false positives. Two unannotated genes are found within these sequences. All genes can be found by using selection and antiselection of features. 29 of the protein coding genes have the /evidence=EXPERIMENTAL qualifier in the EMBL feature table.

The table below gives the results of GeneFinding in these Test_Sequences


Gene symbol/Name			access.	spliced	introns	agrees? 
================			======	=======	=======	=======


PRH1 ATP-dependant RNA Helicase		D13249	yes	2 2	+
dsk1+					D13447	no		+
gamma glutamylcysteine synthase		D55676	yes	1 0	- 	first exon missing; although evidence > blastx homology 
rpb6					L00597	yes	1 1	+
cdc8					L04126	yes	1 1	-	uses downstream ATG
pold					L07734	yes	1 1 +1	- 	splice AT^G to A > ATA , bug!
exo2					L35232	yes	2 1	-	uses rare donor gttgtt
heat shock protein (sis1;Psi)		L37753	no		+	another gene at 1897 2133
fus1					L37838	no	    +1	- 	intron internal to orf
cdc27+                         		M74062	yes	5 5	+
cdc27+ mRNA				M83307	n/a		+
let1					U02280	yes	1 1 +1	-	extra exon
cki1 mRNA				U06929	n/a		+
HIS1 mRNA				U07830	n/a		+
HIS5 mRNA				U07831	n/a		+
cnx1					U13389	n/a		+
ENO1 mRNA				U13799 	no		+
rpb1					X56564	yes	6 6	+
rad9					X58231	yes	3 3	+
SSP1					X59987	no		+
sts1+					X63549	no	    +1	-	extra exon
vma1+					X68580	yes	2 2	+
vma2					X69638	yes	4 4	+
FIB					X69930	no		+	another gene at -3088 -1799
rad26					X76558	yes	2 2	+
gar2					Z48166	no		+
sak1 					U19978	no		+
IDI1					U21154	no		+
chk1					L13742	yes	6 6	+
mcs2					S59895	yes	2 2	+
csk1					S59896	yes 	2 2	+
rad1					M38132	no	    +1	-	extra exon

--------------------------------------------------------------------------------------------------------------------------------

								32 Total
								24 Exact predictions	
								16 Spliced gene prediction	
								11 Exact spliced predictions
					introns		41 39	
					false		     +5 


GeneFinder parameters

Feature parameters

Features range                  10000 
3' splice cutoff                2.00	
5' splicecutoff                 1.00
ATG cutoff                      0.00

Autofind parameters

min intron length               30
min exon length                 3
intron cost                    -4.5
intron rate per log bp         -2.0
coding:intron score ratio       1.00


GeneFinder_Tutorial.user_segments were included for each prediction
using Read Segments

Correspondance to Sean Walsh svw@sanger.ac.uk


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.00 ATG cutoff 0.00 Autofind parameters min intron length 30 min exon length 3 intron cost step2.html000664 022642 001631 00000003346 06505776233 013441 0ustar00valYeasties000000 000000 Feature parameters

Feature parameters

Bring up the GeneFinder menu (under the GeneFind.. button) and select [Feature Parameters]. Change the parameters to the ones given below and press the OK button.

Each Genefinder Feature is given a score. Click on an ATG, splice donor (Splice5) or splice acceptor (Splice3) and look at the blue information bar at the top of the screen to see the score. The Feature parameters are the minimum score for a feature to be included on the fmap.

The score of genefinder features is calculated from weight matrices (ATG, donor and acceptor) and codon usage tables in the wgf/ directory of ACEDB

To recalculate the GeneFinder Features click on the GeneFind.. button with the left mouse button OR bring up the GeneFinder menu with the right mouse button and select [GeneFinder Features]. Note there are fewer splice sites marked on the fmap.

The GeneFinder Features used for the last gene prediction are still highlighted in green. Press the Clear button at the top of the screen, otherwise GeneFinder will only use these features and subsequently find the same gene.

Choose the [AutoFind Gene] option from the GeneFinder menu and press Continue when the dialogue box appears. The old temp_gene is overwritten with a new prediction (see below).

Go to Step 3


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Press the Clear button at the top of the screen, otherwise GeneFinder will only use these features and subsequently find the same gene.

Choose the [AutoFind Gene] option from the GeneFinder menu and press Continue when the dialogue box appears. Thestep3.html000664 022642 001631 00000004624 06505776233 013442 0ustar00valYeasties000000 000000 AutoFind Parameters

AutoFind Parameters

Bring up the GeneFinder menu (under the GeneFind.. button) and select [AutoFind Parameters]. Change the parameters to the ones given below and press the OK button.

AutoFind parameters are weights and limits which are used by the GeneFinder algorithm to predict a gene. AutoFind parameters can be varied by the user, which allows GeneFinder to be tuned to find genes typical of a particular organism; in this case Schizosaccharomyces pombe

AutoFind parameters are:

min intron length

min exon length does not apply to the first and last exon

intron cost the cost to the gene score for including each intron

intron rate per log bp an additional cost for including an intron above a certain length is added according to this weight (see Aside)

coding:intron score ratio the coding score is derived from a codon usage table and visualised on the fmap as blocks of coding potential. The coding score is increased proportionately by this weight.

[Aside: the additional cost is added above 100 bp (given a minimum intron length of 30 bp). This is hard coded in ACEDB 4.1 (and has been modified for this database) but will become an AutoFind parameter in the next release. The additional cost is the log of the additional length over 100bp multiplied by the weight "intron rate per log bp".]

Press the Clear button to remove the green highlighting of GeneFinder features and then select [AutoFind Gene] under the GeneFind.. menu. Press Continue in the dialouge box which appears. The temp_gene is overwritten. Note that the GeneFinder prediction exactly matches the gene specified in the EMBL record. Double click the text EM:D13249 to the left of the scale and inspect the full text of the EMBL record. You will see that the intron/exon structure has been confirmed experimentally.

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ue in the dialouge box which appears. The temp_gene is overwritten. Note that the GeneFinder prediction exacstep4.html000664 022642 001631 00000002311 06505776233 013432 0ustar00valYeasties000000 000000 Show Selected

Show Selected

Double click the Test_Sequence EM:D55676 from the KeySet. With GeneFinder Features and AutoFind Parameters set as in Steps 2 and 3, choose [GeneFinder Features] and then [AutoFind gene].

The temp_gene does not have the same structure as the gene from the EMBL record (see below).

Inspect the gene score of the temp_gene by chosing the [Show Selected] option in the GeneFind.. menu. The following table should appear:

Now press Clear and click on the other gene. Choose [Gene -> Selected] from the GeneFind.. menu and then [Show Selected]. The table below should appear. The temp_gene was chosen in preferance to the other gene due to its larger combined exon score, whilst combined intron scores were approximately equivalent.

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> Now press Clear and click on the other gene. Choose [Gene -> Selected] from the GeneFind.. menu and then [Show Selected]. The table below should appear. The temp_gene was chosen in preferance to the other gene due to its larger combined exon score, whilst combined intron scores werstep5.html000664 022642 001631 00000002561 06505776233 013442 0ustar00valYeasties000000 000000 Select and Antiselect

Select and Antiselect

The exon/intron structure of the gene in the EM:D55676 EMBL record has been experimentally determined. BlastX homology to another gamma-glutamylcysteine synthetase from Rattus norvegicus confirms this structure (click on the BlastX boxes to see this).

To predict this gene with GeneFinder it would be possible to modify AutoFind Parameters until it was found. However, GeneFinder can be forced to use or disregard selected features. To do this, press the Clear button to remove all highlighting of features. Select the ATG (base 684-686)(see image below) with the right mouse button and a menu appears. Choose [Select] from the menu and the ATG is highlighted in green. Now select the splice donor (base 1034-1035) with the right mouse button and choose [Antiselect]. The donor is hightlighted in pale green. From the GeneFind.. menu choose [AutoFind gene]. The gene prediction is the same as that in the EMBL record.

Note that Unselect is also available from the feature selection menu.

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select the splice donor (base 1034-1035) with the right mouse button and choose [Antiselect]. The donor is hightlighted in pale green. From thstep6.html000664 022642 001631 00000003074 06505776234 013444 0ustar00valYeasties000000 000000 Read Segments

Read Segments

GeneFinder does not directly take into account splice branch sites when predicting genes. However it is possible to give GeneFinder branch site information by supplying splice acceptor sites which are a combination of splice branch and acceptor. The combined branch/acceptor motifs are located in the sequence by a program called Sp3splice which is external to ACEDB, and the results are written to a file with the ".useg" extension. Note that "useg" is a contraction of User Segments. The following example is a demonstration of the value of including splice branch information for predicting genes in S. pombe

Bring up the fmap for Test_Sequence EM:X56564 and choose [GeneFinder features] followed by [AutoFind gene] from the GeneFind.. menu. The predicted gene is not the same as the gene from the EMBL record (see below).

Select [GeneFinder parameters] from the GeneFind.. menu and change 3-splice cutoff to 2.00 (see below). Splice acceptors calculated by GeneFinder do not attain scores over 2.00, so this ensures that they are not used. Now select [GeneFinder features] from the GeneFind.. menu and notice that all the acceptors have disappeared from the fmap.

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elect [GeneFinder parameters] from the GeneFind.. menu and change 3-splice cutoff to 2.00 (see below). Splice acceptors calculated by GeneFinder do not attain scores over 2.00, so this ensures that they are not used. Now select [GeneFinder features] from the GeneFind.. menu and notice that all the acceptors have disappeared from the fmap.

Go to Step 7


Read Segments

Read Segments Continued

With the right mouse button, pick the white background of the fmap and a menu appears; choose [Read Segments]. A file chooser appears. Move into the directory usegs/, select the file X56564 and press the OK button. A message box appears with the number of segments added; press Continue. Note that acceptors have appeared. These are a combination of splice branch and acceptor sequences. In the image below a combined splice branch/acceptor is highlighted in pink.

Press the Clear button. Choose [AutoFind gene] from the GeneFind.. menu. The predicted gene is now the same as the gene from the EMBL record (see below).

Go to Step 8


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ion of splice branch and acceptor sequences. In the image below a combined splice branch/acceptor is highlighted in pink.

Press the Clear button. Choose [AutoFind gene] from the GeneFind.. menu. The predicted gene is now the same as the gene from the EMBL record (see below).

Go to Step 8


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Other GeneFinding Controls

Other GeneFinder Controls

GeneFinder predictions can be saved as Sequence objects by chosing [Fix temp_gene] from the GeneFind.. menu. Genes can be removed by picking a gene on the fmap and selecting [Remove Gene]. Both of these operations require write access to the database.

Gene predictions can be dumped into an EMBL feature table by selecting the [EMBL dump] option from the menu which is activated by picking the white fmap background. The option [Import Sequence] from the same menu allows a sequence file (in plain or FASTA format) to be read into the fmap. Both these operations do not require write access.

An example of an EMBL dump is given below. Note that the /organism identifier is C. elegans by default.

XX
FH   Key             Location/Qualifiers
FH
FT   source          1..7079
FT                   /organism="Caenorhabditis elegans"
FT                   /clone="EM:X56564"
FT   CDS             join(911..925,961..986,
FT                   1027..1099,1139..1214,1260..1319,
FT                   1412..1518,1566..6467)
FT                   /gene="rpb1"
FT   CDS             join(911..925,961..986,
FT                   1027..1099,1139..1214,1260..1319,
FT                   1412..1518,1566..6467)
FT                   /product="temp_gene"
XX
SQ   Sequence  7079 BP;   2024 A; 1386 C; 1394 G; 2275 T; 0 other;
     aagcttctag agccactcct gacattaaag aacagcatgc taagaaaccg aagcgaaagc
     acacccgttc aacagtacct acaagcaatg ttgaaccagt ttctcaacct caaccctctc
     ctgacaaaat cgtttctagc cccaatccgc catcagcgaa aagggaaaaa aagaaacgtc
     gcaaaagctc gatgtcttct tccattacca ......etc.

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T /product="temp_gene" XX SQ Sequence 7079 BP; 2024 A; 1386 C; 1394 G; 2step9.html000664 022642 001631 00000001432 06505776234 013443 0ustar00valYeasties000000 000000 Active Zone

Active Zone

GeneFinder always finds the highest scoring gene for a given set of AutoFind parameters. In sequences which contain more than one gene it will be necessary to limit the region in which GeneFinder looks in order to exclude the highest scoring gene once it has been found. This is done by entering co-ordinates in the Active Zone box. The blue line on the Summary bar (yellow) denotes the region which is excluded. In the example below a second gene is found within the sequence.

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e it will be necessary to limit the region in which GeneFinder looks in order to exclude the highest scoring gene once it has been found. This is done by entering co-ordinates in the Active Zone box. 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