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<HTML>
<HEAD>
<TITLE>
EMBOSS: est2genome
</TITLE>
</HEAD>
<BODY BGCOLOR="#FFFFFF" text="#000000">
<table align=center border=0 cellspacing=0 cellpadding=0>
<tr><td valign=top>
<A HREF="/" ONMOUSEOVER="self.status='Go to the EMBOSS home page';return true"><img border=0 src="emboss_icon.jpg" alt="" width=150 height=48></a>
</td>
<td align=left valign=middle>
<b><font size="+6">
est2genome
</font></b>
</td></tr>
</table>
<br>
<p>
<H2>
Function
</H2>
Align EST and genomic DNA sequences
<H2>
Description
</H2>
<b>est2genome</b> is a software tool to aid the prediction of genes by
sequence homology. The program will align a set of spliced nucleotide
sequences (ESTs cDNAs or mRNAs) to an unspliced genomic DNA sequence,
inserting introns of arbitrary length when needed. In addition, where
feasible introns start and stop at the splice consensus dinucleotides
GT and AG.
<p>
Unless instructed otherwise, the program makes three alignments: First
it compares both stands of the spliced sequence against the forward
strand of the genomic, assuming the splice consensus GT/AG (ie in the
forward gene direction). The maximum-scoring orientation is then
realigned assuming the splice consensus CT/AC (ie in the reversed gene
direction). Only the overall maximum-scoring alignment is reported.
<p>
The program outputs a list of the exons and introns it has found. The
format is like that of MSPcrunch, ie a list of matching segments. This
format is easy to parse into other software. The program also
indicates, based on the splice site information, the gene's predicted
direction of transcription. Optionally the full sequence alignment is
printed as well (see the example).
<H2>
Algorithm
</H2>
The program uses a linear-space divide-and-conquer strategy (Myers and
Miller, 1988; Huang, 1994) to limit memory use:
<p>
1. A first pass Smith-Waterman local alignment scan is done to find the
start and end of the maximally scoring segments.
<p>
2. Subsequences corresponding to these segments are extracted
<p>
3a. If the product of the subsequences' lengths is less than a
user-defined threshold (i.e. they will fit in memory) the segments are
realigned using the Needleman-Wunsch global alignment algorithm, which
will give the same result as the Smith-Waterman since the subsequences
are guaranteed to align end-to-end.
<p>
3b. If the product of the lengths exceeds the threshold (a full
alignment will not fit in memory) the alignment is made recursively by
splitting the spliced (EST) sequence in half and finding the genome
sequence position which aligns with the mid-point. The process is
repeated until the product of gthe lengths is less than the threshold.
The divided sequences are aligned separately and then merged.
<p>
4. The genome sequence is searched against the forward and reverse
strands of the spliced (EST) sequence, assuming a forward gene splicing
direction (i.e. GT/AG consensus).
<p>
5. Then the best-scoring orientation is realigned assuming reverse
splicing (CT/AC consensus). The overall best alignment is reported.
<H2>
Usage
</H2>
<b>Here is a sample session with est2genome</b>
<p>
<p>
<table width="90%"><tr><td bgcolor="#CCFFFF"><pre>
% <b>est2genome </b>
Align EST and genomic DNA sequences
Spliced EST nucleotide sequence(s): <b>tembl:h45989</b>
Unspliced genomic nucleotide sequence: <b>tembl:z69719</b>
Output file [h45989.est2genome]: <b></b>
</pre></td></tr></table><p>
<p>
<a href="#input.1">Go to the input files for this example</a><br><a href="#output.1">Go to the output files for this example</a><p><p>
<H2>
Command line arguments
</H2>
<table CELLSPACING=0 CELLPADDING=3 BGCOLOR="#f5f5ff" ><tr><td>
<pre>
Standard (Mandatory) qualifiers:
[-estsequence] seqall Spliced EST nucleotide sequence(s)
[-genomesequence] sequence Unspliced genomic nucleotide sequence
[-outfile] outfile [*.est2genome] Output file name
Additional (Optional) qualifiers:
-match integer [1] Score for matching two bases (Any
integer value)
-mismatch integer [1] Cost for mismatching two bases (Any
integer value)
-gappenalty integer [2] Cost for deleting a single base in
either sequence, excluding introns (Any
integer value)
-intronpenalty integer [40] Cost for an intron, independent of
length. (Any integer value)
-splicepenalty integer [20] Cost for an intron, independent of
length and starting/ending on donor-acceptor
sites (Any integer value)
-minscore integer [30] Exclude alignments with scores below
this threshold score. (Any integer value)
Advanced (Unprompted) qualifiers:
-reverse boolean Reverse the orientation of the EST sequence
-[no]splice boolean [Y] Use donor and acceptor splice sites. If
you want to ignore donor-acceptor sites then
set this to be false.
-mode menu [both] This determines the comparion mode.
The default value is 'both', in which case
both strands of the est are compared
assuming a forward gene direction (ie GT/AG
splice sites), and the best comparsion
redone assuming a reversed (CT/AC) gene
splicing direction. The other allowed modes
are 'forward', when just the forward strand
is searched, and 'reverse', ditto for the
reverse strand. (Values: both (Both
strands); forward (Forward strand only);
reverse (Reverse strand only))
-[no]best boolean [Y] You can print out all comparisons
instead of just the best one by setting this
to be false.
-space float [10.0] For linear-space recursion. If
product of sequence lengths divided by 4
exceeds this then a divide-and-conquer
strategy is used to control the memory
requirements. In this way very long
sequences can be aligned.
If you have a machine with plenty of memory
you can raise this parameter (but do not
exceed the machine's physical RAM) (Any
numeric value)
-shuffle integer [0] Shuffle (Any integer value)
-seed integer [20825] Random number seed (Any integer
value)
-align boolean Show the alignment. The alignment includes
the first and last 5 bases of each intron,
together with the intron width. The
direction of splicing is indicated by angle
brackets (forward or reverse) or ????
(unknown).
-width integer [50] Alignment width (Any integer value)
Associated qualifiers:
"-estsequence" associated qualifiers
-sbegin1 integer Start of each sequence to be used
-send1 integer End of each sequence to be used
-sreverse1 boolean Reverse (if DNA)
-sask1 boolean Ask for begin/end/reverse
-snucleotide1 boolean Sequence is nucleotide
-sprotein1 boolean Sequence is protein
-slower1 boolean Make lower case
-supper1 boolean Make upper case
-sformat1 string Input sequence format
-sdbname1 string Database name
-sid1 string Entryname
-ufo1 string UFO features
-fformat1 string Features format
-fopenfile1 string Features file name
"-genomesequence" associated qualifiers
-sbegin2 integer Start of the sequence to be used
-send2 integer End of the sequence to be used
-sreverse2 boolean Reverse (if DNA)
-sask2 boolean Ask for begin/end/reverse
-snucleotide2 boolean Sequence is nucleotide
-sprotein2 boolean Sequence is protein
-slower2 boolean Make lower case
-supper2 boolean Make upper case
-sformat2 string Input sequence format
-sdbname2 string Database name
-sid2 string Entryname
-ufo2 string UFO features
-fformat2 string Features format
-fopenfile2 string Features file name
"-outfile" associated qualifiers
-odirectory3 string Output directory
General qualifiers:
-auto boolean Turn off prompts
-stdout boolean Write standard output
-filter boolean Read standard input, write standard output
-options boolean Prompt for standard and additional values
-debug boolean Write debug output to program.dbg
-verbose boolean Report some/full command line options
-help boolean Report command line options. More
information on associated and general
qualifiers can be found with -help -verbose
-warning boolean Report warnings
-error boolean Report errors
-fatal boolean Report fatal errors
-die boolean Report dying program messages
</pre>
</pre>
</td></tr></table>
<P>
<table border cellspacing=0 cellpadding=3 bgcolor="#ccccff">
<tr bgcolor="#FFFFCC">
<th align="left" colspan=2>Standard (Mandatory) qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
</tr>
<tr>
<td>[-estsequence]<br>(Parameter 1)</td>
<td>Spliced EST nucleotide sequence(s)</td>
<td>Readable sequence(s)</td>
<td><b>Required</b></td>
</tr>
<tr>
<td>[-genomesequence]<br>(Parameter 2)</td>
<td>Unspliced genomic nucleotide sequence</td>
<td>Readable sequence</td>
<td><b>Required</b></td>
</tr>
<tr>
<td>[-outfile]<br>(Parameter 3)</td>
<td>Output file name</td>
<td>Output file</td>
<td><i><*></i>.est2genome</td>
</tr>
<tr bgcolor="#FFFFCC">
<th align="left" colspan=2>Additional (Optional) qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
</tr>
<tr>
<td>-match</td>
<td>Score for matching two bases</td>
<td>Any integer value</td>
<td>1</td>
</tr>
<tr>
<td>-mismatch</td>
<td>Cost for mismatching two bases</td>
<td>Any integer value</td>
<td>1</td>
</tr>
<tr>
<td>-gappenalty</td>
<td>Cost for deleting a single base in either sequence, excluding introns</td>
<td>Any integer value</td>
<td>2</td>
</tr>
<tr>
<td>-intronpenalty</td>
<td>Cost for an intron, independent of length.</td>
<td>Any integer value</td>
<td>40</td>
</tr>
<tr>
<td>-splicepenalty</td>
<td>Cost for an intron, independent of length and starting/ending on donor-acceptor sites</td>
<td>Any integer value</td>
<td>20</td>
</tr>
<tr>
<td>-minscore</td>
<td>Exclude alignments with scores below this threshold score.</td>
<td>Any integer value</td>
<td>30</td>
</tr>
<tr bgcolor="#FFFFCC">
<th align="left" colspan=2>Advanced (Unprompted) qualifiers</th>
<th align="left">Allowed values</th>
<th align="left">Default</th>
</tr>
<tr>
<td>-reverse</td>
<td>Reverse the orientation of the EST sequence</td>
<td>Boolean value Yes/No</td>
<td>No</td>
</tr>
<tr>
<td>-[no]splice</td>
<td>Use donor and acceptor splice sites. If you want to ignore donor-acceptor sites then set this to be false.</td>
<td>Boolean value Yes/No</td>
<td>Yes</td>
</tr>
<tr>
<td>-mode</td>
<td>This determines the comparion mode. The default value is 'both', in which case both strands of the est are compared assuming a forward gene direction (ie GT/AG splice sites), and the best comparsion redone assuming a reversed (CT/AC) gene splicing direction. The other allowed modes are 'forward', when just the forward strand is searched, and 'reverse', ditto for the reverse strand.</td>
<td><table><tr><td>both</td> <td><i>(Both strands)</i></td></tr><tr><td>forward</td> <td><i>(Forward strand only)</i></td></tr><tr><td>reverse</td> <td><i>(Reverse strand only)</i></td></tr></table></td>
<td>both</td>
</tr>
<tr>
<td>-[no]best</td>
<td>You can print out all comparisons instead of just the best one by setting this to be false.</td>
<td>Boolean value Yes/No</td>
<td>Yes</td>
</tr>
<tr>
<td>-space</td>
<td>For linear-space recursion. If product of sequence lengths divided by 4 exceeds this then a divide-and-conquer strategy is used to control the memory requirements. In this way very long sequences can be aligned.
If you have a machine with plenty of memory you can raise this parameter (but do not exceed the machine's physical RAM)</td>
<td>Any numeric value</td>
<td>10.0</td>
</tr>
<tr>
<td>-shuffle</td>
<td>Shuffle</td>
<td>Any integer value</td>
<td>0</td>
</tr>
<tr>
<td>-seed</td>
<td>Random number seed</td>
<td>Any integer value</td>
<td>20825</td>
</tr>
<tr>
<td>-align</td>
<td>Show the alignment. The alignment includes the first and last 5 bases of each intron, together with the intron width. The direction of splicing is indicated by angle brackets (forward or reverse) or ???? (unknown).</td>
<td>Boolean value Yes/No</td>
<td>No</td>
</tr>
<tr>
<td>-width</td>
<td>Alignment width</td>
<td>Any integer value</td>
<td>50</td>
</tr>
</table>
<H2>
Input file format
</H2>
<b>est2genome</b> reads two nucleotide sequences. The first is an EST
sequence (a single read or a finished cDNA). The second is a genomic
finished sequence.
<p>
<a name="input.1"></a>
<h3>Input files for usage example </h3>
'tembl:h45989' is a sequence entry in the example nucleic acid database 'tembl'
<p>
<p><h3>Database entry: tembl:h45989</h3>
<table width="90%"><tr><td bgcolor="#FFCCFF">
<pre>
ID H45989; SV 1; linear; mRNA; EST; HUM; 495 BP.
XX
AC H45989;
XX
DT 18-NOV-1995 (Rel. 45, Created)
DT 04-MAR-2000 (Rel. 63, Last updated, Version 2)
XX
DE yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone
DE IMAGE:177794 3', mRNA sequence.
XX
KW EST.
XX
OS Homo sapiens (human)
OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
OC Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae;
OC Homo.
XX
RN [1]
RP 1-495
RA Hillier L., Clark N., Dubuque T., Elliston K., Hawkins M., Holman M.,
RA Hultman M., Kucaba T., Le M., Lennon G., Marra M., Parsons J., Rifkin L.,
RA Rohlfing T., Soares M., Tan F., Trevaskis E., Waterston R., Williamson A.,
RA Wohldmann P., Wilson R.;
RT "The WashU-Merck EST Project";
RL Unpublished.
XX
DR GDB; 3839990.
DR GDB; 4193257.
DR RZPD; IMAGp998F03326.
DR UNILIB; 555; 300.
XX
CC On May 8, 1995 this sequence version replaced gi:800819.
CC Contact: Wilson RK
CC Washington University School of Medicine
CC 4444 Forest Park Parkway, Box 8501, St. Louis, MO 63108
CC Tel: 314 286 1800
CC Fax: 314 286 1810
CC Email: [email protected]
CC Insert Size: 544
CC High quality sequence stops: 265
CC Source: IMAGE Consortium, LLNL
CC This clone is available royalty-free through LLNL ; contact the
CC IMAGE Consortium ([email protected]) for further information.
CC Possible reversed clone: polyT not found
CC Insert Length: 544 Std Error: 0.00
CC Seq primer: SP6
CC High quality sequence stop: 265.
XX
FH Key Location/Qualifiers
FH
FT source 1..495
FT /organism="Homo sapiens"
FT /lab_host="DH10B (ampicillin resistant)"
FT /mol_type="mRNA"
FT /sex="Male"
FT /dev_stage="55-year old"
FT /clone_lib="Soares adult brain N2b5HB55Y"
FT /clone="IMAGE:177794"
FT /note="Organ: brain; Vector: pT7T3D (Pharmacia) with a
FT modified polylinker; Site_1: Not I; Site_2: Eco RI; 1st
FT strand cDNA was primed with a Not I - oligo(dT) primer [5'
FT TGTTACCAATCTGAAGTGGGAGCGGCCGCGCTTTTTTTTTTTTTTTTTTT 3'],
FT double-stranded cDNA was size selected, ligated to Eco RI
FT adapters (Pharmacia), digested with Not I and cloned into
FT the Not I and Eco RI sites of a modified pT7T3 vector
FT (Pharmacia). Library went through one round of
FT normalization to a Cot = 53. Library constructed by Bento
FT Soares and M.Fatima Bonaldo. The adult brain RNA was
FT provided by Dr. Donald H. Gilden. Tissue was acquired 17-18
FT hours after death which occurred in consequence of a
FT ruptured aortic aneurysm. RNA was prepared from a pool of
FT tissues representing the following areas of the brain:
FT frontal, parietal, temporal and occipital cortex from the
FT left and right hemispheres, subcortical white matter, basal
FT ganglia, thalamus, cerebellum, midbrain, pons and medulla."
FT /db_xref="taxon:9606"
FT /db_xref="RZPD:IMAGp998F03326"
FT /db_xref="UNILIB:555"
XX
SQ Sequence 495 BP; 73 A; 135 C; 169 G; 104 T; 14 other;
ccggnaagct cancttggac caccgactct cgantgnntc gccgcgggag ccggntggan 60
aacctgagcg ggactggnag aaggagcaga gggaggcagc acccggcgtg acggnagtgt 120
gtggggcact caggccttcc gcagtgtcat ctgccacacg gaaggcacgg ccacgggcag 180
gggggtctat gatcttctgc atgcccagct ggcatggccc cacgtagagt ggnntggcgt 240
ctcggtgctg gtcagcgaca cgttgtcctg gctgggcagg tccagctccc ggaggacctg 300
gggcttcagc ttcccgtagc gctggctgca gtgacggatg ctcttgcgct gccatttctg 360
ggtgctgtca ctgtccttgc tcactccaaa ccagttcggc ggtccccctg cggatggtct 420
gtgttgatgg acgtttgggc tttgcagcac cggccgccga gttcatggtn gggtnaagag 480
atttgggttt tttcn 495
//
</pre>
</td></tr></table><p>
<p><h3>Database entry: tembl:z69719</h3>
<table width="90%"><tr><td bgcolor="#FFCCFF">
<pre>
ID Z69719; SV 1; linear; genomic DNA; STD; HUM; 33760 BP.
XX
AC Z69719;
XX
DT 26-FEB-1996 (Rel. 46, Created)
DT 18-JAN-2007 (Rel. 90, Last updated, Version 6)
XX
DE Human DNA sequence from clone XX-CNFG9 on chromosome 16
XX
KW C16orf33; HTG; POLR3K; RHBDF1.
XX
OS Homo sapiens (human)
OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
OC Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae;
OC Homo.
XX
RN [1]
RP 1-33760
RA Kershaw J.;
RT ;
RL Submitted (16-JAN-2007) to the EMBL/GenBank/DDBJ databases.
RL Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK.
RL E-mail enquiries: [email protected] Clone requests:
XX
DR EMBL-JOIN; Z69720.
DR GDB; 11502921.
XX
CC -------------- Genome Center
CC Center: Wellcome Trust Sanger Institute
CC Center code: SC
CC Web site: http://www.sanger.ac.uk
CC Contact: [email protected]
CC --------------
CC
CC This sequence was finished as follows unless otherwise noted: all regions
CC were either double-stranded or sequenced with an alternate chemistry or
CC covered by high quality data (i.e., phred quality >= 30); an attempt was
CC made to resolve all sequencing problems, such as compressions and repeats;
CC all regions were covered by at least one subclone; and the assembly was
CC confirmed by restriction digest, except on the rare occasion of the clone
CC being a YAC.
CC
CC The following abbreviations are used to associate primary accession
CC numbers given in the feature table with their source databases:
CC Em:, EMBL; Sw:, SWISSPROT; Tr:, TREMBL; Wp:, WORMPEP;
CC Information on the WORMPEP database can be found at
CC http://www.sanger.ac.uk/Projects/C_elegans/wormpep
XX
FH Key Location/Qualifiers
<font color=red> [Part of this file has been deleted for brevity]</font>
gagacagcag agtgctcagc tcatgaagga ggcaccagcc gccatgcctc tacatccagg 30840
tctcctgggg ttcccacctc cacaaaaacc cccactgcta ggagtgcagg caggagggga 30900
cctgagaacc gacagttata ggtcctgcgg gtgggcagtg ctgggtgttc tggtctgccc 30960
cacccctgtg tgcctagatc cccatctggg cctcaagtgg gtgggattcc aaaggaagag 31020
ccggagtagg cgtggggagg ggcaggccca ggctggacaa agagtctggc cagggagcgg 31080
cacattgccc tcccagagac agtggctcag tgtccaggcc ttccccaggc gcacagtggg 31140
ctcttgttcc cagaaagccc ctcgggggga tccaaacagt gtctccccca ccccgctgac 31200
ccctcagtgt atggggaaac cgtggcccac ggaaggcctc actgcctggg gtcacacagc 31260
atctgagtca ctgcagcagc ctcacagctg ccagcccagg cccagcccca tcaggagaca 31320
cccaaagcca cagtgcatcc caggaccagc tgggggggct gcgggcagga ctctcgatga 31380
ggctgaggga cgaggagggt caagggagcc actggcgcca tgcatgctga cgtcccctct 31440
ggctgcctgc agagcctggt gtggaagggc tgagtggggg atggtggaga gtcctgttaa 31500
ctcaggtttc tgctctgggg atgtctgggc acccatcaag ctggccgcgt gcacaggtgc 31560
agggagagcc agaaagcagg agccgatgca gggaggccac tggggacagc ccaggctgat 31620
gcttgggccc catgtgtctc caccacctac aaccctaagc aagcctcagc tttcccatct 31680
ggaaatcagg ggtcacagca gtgcctggca cagtagcagc ggctgactcc atcacagggt 31740
ggtgtagcct gtgggtactt ggcactctct gaggggcagg agctgggggg tgaaaggacc 31800
ctagagcata tgcaacaaga gggcagccct ggggacacct ggggacagaa ccctccaaag 31860
gtgtcgagtt tgggaagaga ctagagagaa gctctggcca gtccaggcat agacagtggc 31920
cacagccagt ggagagctgc atcctcaggt gtgagcagca accacctctg tactcaggcc 31980
tgccctgcac actcacagga ccatgctggc agggacaact ggcggcggag ttgactgcca 32040
accccggggc cagaaccatc aagcctgggc tctgctccgc ccaaggaact gcctgctgcc 32100
gaggtcagct ggagcaaggg gcctcacccc gggacacctt cccagacgtg tcctcagctc 32160
acatgagcct catcccaggg ggatgtggct cctccagcat ccccacccac acgctgctct 32220
ctgaccctca gtcttctgtt tgactcctaa tctgaagctc aatcctagat ctcccttgag 32280
aagggggtca ccagctgtct ggcagcccag cctccaggtc ttctggatta atgaagggaa 32340
agtcacctgg cctctctgcc ttgtctatta atggcatcat gctgagaatg atatttgcta 32400
ggccctttgc aaaccccaaa gtgctcttca accctcccag tgaagcctct tcttttctgt 32460
ggaagaaatg aggttcaggg tggagcaggg caggcctgag acctttgcag ggttctctcc 32520
aggtccccag caggacagac tggcaccctg cctcccctca tcaccctaga caaggagaca 32580
gaacaagagg ttccctgcta caggccatct gtgagggaag ccgccctagg gcctgtagac 32640
acaggaatcc ctgaggacct gacctgtgag ggtagtgcac aaaggggcca gcacttggca 32700
ggaggggggg gggcactgcc ccaaggctca gctagcaaat gtggcacagg ggtcaccaga 32760
gctaaacccc tgactcagtt gggtctgaca ggggctgaca tggcagacac acccaggaat 32820
caggggacac caagtgcagc tcagggcacc tgtccaggcc acacagtcag aaaggggatg 32880
gcagcaagga cttagctaca ctagattctg ggggtaaact gcctggtatg ctggtcactg 32940
ctagtcccca gtctggagtc tagctgggtc tcaggagtta ggcgaaaaca ccctccccag 33000
gctgcaggtg ggagaggccc acatcccctg cacacgtctg gccagaggac agatgggcag 33060
cccagtcacc agtcagagcc ctccagaggt gtccctgact gaccctacac acatgcaccc 33120
aggtgcccag gcacccttgg gctcagcaac cctgcaaccc cctcccagga cccaccagaa 33180
gcaggatagg actagagagg ccacaggagg gaaaccaagt cagagcagaa atggcttcgg 33240
tcctcagcag cctggctcag cttcctcaaa ccagatcctg actgatcaca ctggtctgtc 33300
taacccctgg gaggggtcct ctgtatccat cttacagata aggaaactga ggctcagaga 33360
agcccatcac tgcctaaggt cccagggcct ataagggagc tcaaagcctt gggccaggtc 33420
tgcccaggag ctgcagtgga agggaccctg tctgcagacc cccagaagac aaggcagacc 33480
acctgggttc ttcagccttg tggctgtgga cggctgtcag acccttctaa gaccccttgc 33540
cacctgctcc atcaggggca tctcagttga agaaggaagg actcaccccc aaaatcgtcc 33600
aactcagaaa aaaaggcaga agccaaggaa tccaatcact gggcaaaatg tgatcctggc 33660
acagacactg aggtggggga actggagccg gtgtggcgga ggccctcaca gccaagagca 33720
actgggggtg ccctgggcag ggactgtagc tgggaagatc 33760
//
</pre>
</td></tr></table><p>
<H2>
Output file format
</H2>
<a name="output.1"></a>
<h3>Output files for usage example </h3>
<p><h3>File: h45989.est2genome</h3>
<table width="90%"><tr><td bgcolor="#CCFFCC">
<pre>
Note Best alignment is between forward est and forward genome, but splice sites imply REVERSED GENE
Exon 163 91.8 25685 25874 Z69719 1 193 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
-Intron -20 0.0 25875 26278 Z69719
Exon 207 98.1 26279 26492 Z69719 194 407 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
-Intron -20 0.0 26493 27390 Z69719
Exon 63 86.4 27391 27476 Z69719 408 494 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Span 393 93.6 25685 27476 Z69719 1 494 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 14 83.3 25685 25702 Z69719 1 18 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 28 85.7 25703 25737 Z69719 20 54 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 4 100.0 25738 25741 Z69719 56 59 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 13 100.0 25742 25754 Z69719 61 73 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 4 100.0 25756 25759 Z69719 74 77 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 110 97.4 25760 25874 Z69719 79 193 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 37 100.0 26279 26315 Z69719 194 230 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 162 98.8 26317 26480 Z69719 231 394 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 12 100.0 26481 26492 Z69719 396 407 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 16 100.0 27391 27406 Z69719 408 423 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 10 91.7 27407 27418 Z69719 425 436 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 19 95.2 27419 27439 Z69719 438 458 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
Segment 24 80.6 27441 27476 Z69719 459 494 H45989 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence.
</pre>
</td></tr></table><p>
<p>
<h3>MSP type segments</h3>
There are four types of segment,
<ol>
<li>each gapped <b>Exon</b>
<li>each <b>Intron</b> (marked with a ? if it does not start GT and end AG)
<li>the complete alignment <b>Span</b>
<li>individual ungapped matching <b>Segments</b>.
</ol>
<p>
The score for <b>Exon</b> segments is the alignment score excluding
flanking intron penalties. The <b>Span</b> score is the total
including the intron costs.
<p>
The coordinates of the genomic sequence always refer to the positive
strand, but are swapped if the est has been reversed. The splice
direction of Introns are indicated as <b>+Intron</b> (forward, splice
sites GT/AG) or <b>-Intron</b> (reverse, splice sites CT/AC), or
<b>?Intron</b> (unknown direction). <b>Segment</b> entries give the
alignment as a series of ungapped matching segments.
<h3>Full alignment</h3>
You get the alignment if the -align switch is set. The alignment
includes the first and last 5 bases of each intron, together with the
intron width. The direction of splicing is indicated by
>>>> (forward) or <<<< (reverse) or ????
(unknown)
<!--
<pre>
HSNFG9 vs HS989235:
HSNFG9 25685 cccggaagctcatcttgg-ccaccgactctcgcttgcgccgccgcgggag 25733
|| | ||||||| ||||| ||||||||||||| || |||||||||||
HS989235 1 ccggnaagctcancttggaccaccgactctcgantgnntcgccgcgggag 50
HSNFG9 25734 ccgg-tgga-aacctgagcgggagctgg-agaaggagcagagggaggcag 25780
|||| |||| ||||||||||||| |||| |||||||||||||||||||||
HS989235 51 ccggntgganaacctgagcggga-ctggnagaaggagcagagggaggcag 99
HSNFG9 25781 cacccggcgtgacgggagtgtgtggggcactcaggccttccgcagtgtca 25830
||||||||||||||| ||||||||||||||||||||||||||||||||||
HS989235 100 cacccggcgtgacggnagtgtgtggggcactcaggccttccgcagtgtca 149
HSNFG9 25831 tctgccacacggaaggcacggccacgggccagggggtctatgatctgga. 25874
||||||||||||||||||||||||||||| |||||||||||||<<<<<
HS989235 150 tctgccacacggaaggcacggccacgggcaggggggtctatgat...... 193
HSNFG9 25874 ....cataccttctgcatgcccagctggcatggccccacgtagagtgggg 26319
404 <<<<<||||||||||||||||||||||||||||||||||||| ||
HS989235 193 .........cttctgcatgcccagctggcatggccccacgtagagt-ggn 233
HSNFG9 26320 gtggcgtctcggtgctggtcagcgacacgttgtcctggctgggcaggtcc 26369
|||||||||||||||||||||||||||||||||||||||||||||||||
HS989235 234 ntggcgtctcggtgctggtcagcgacacgttgtcctggctgggcaggtcc 283
HSNFG9 26370 agctcccggaggacctggggcttcagcttcccgtagcgctggctgcagtg 26419
||||||||||||||||||||||||||||||||||||||||||||||||||
HS989235 284 agctcccggaggacctggggcttcagcttcccgtagcgctggctgcagtg 333
HSNFG9 26420 acggatgctcttgcgctgccatttctgggtgctgtcactgtccttgctca 26469
||||||||||||||||||||||||||||||||||||||||||||||||||
HS989235 334 acggatgctcttgcgctgccatttctgggtgctgtcactgtccttgctca 383
HSNFG9 26470 ctccaaaccag-tcggcggtccccctggc.....ggtacctgcggatggt 27401
||||||||||| ||||||||||||<<<<< 898 <<<<<|||||||||||
HS989235 384 ctccaaaccagttcggcggtcccc...............ctgcggatggt 418
HSNFG9 27402 ctgtg-tgatggacgtct-ggcgttgcagcaccggccgccggagctcatg 27449
||||| |||||||||| | ||| ||||||||||||||||| ||| |||||
HS989235 419 ctgtgttgatggacgtttgggctttgcagcaccggccgcc-gagttcatg 467
HSNFG9 27450 gtggggtgaagagatgtgggctgtctc 27476
|| |||| ||||||| |||| | | ||
HS989235 468 gtngggtnaagagatttgggttttttc 494
Alignment Score: 393
</pre>
In this example there are two reverse introns, of lengths 404 and 898 bp.
-->
<H2>
Data files
</H2>
None
<H2>
Notes
</H2>
<b>est2genome</b> uses a linear-space dynamic-programming
algorithm. It has the following parameters:
<pre>
parameter default description
match 1 score for matching two bases
mismatch 1 cost for mismatching two bases
gap_penalty 2 cost for deleting a single base in
either sequence,
excluding introns
intron_penalty 40 cost for an intron, independent of
length.
splice_penalty 20 cost for an intron, independent of
length and starting/ending on
donor-acceptor sites.
space 10 Space threshold (in megabytes)
for linear-space recursion. If the
product of the two sequence
lengths divided by 4 exceeds this then
a divide-and-conquer strategy is used
to control the memory requirements.
In this way very long sequences can
be aligned.
If you have a machine with plenty of
memory you can raise this parameter
(but do not exceed the machine's
physical RAM)
However, normally you should not need
to change this parameter.
</pre>
There is no gap initiation cost for short gaps, just a penalty
proportional to the length of the gap. Thus the cost of inserting a
gap of length L in the EST is <pre> L*gap_penalty </pre> and the cost
in the genome is
<pre>
min { L*gap_penalty, intron_penalty } or
min { L*gap_penalty, splice_penalty } if the gap starts with GT and ends with AG
(or CT/AC if splice direction reversed)
</pre>
Introns are not allowed in the EST. The difference between the
intron_penalty and splice_penalty allows for some slack in marking the
intron end-points. It is often the case that the best intron
boundaries, from the point of view of minimising mismatches, will not
coincide exactly with the splice consensus, so provided the difference
between the intron/splice penalties outweighs the extra mismatch/indel
costs the alignment will respect the proper boundaries. If the
alignment still prefers boundaries which don't start and end with the
splice consensus then this may indicate errors in the sequences.
<p> The default parameters work well, except for very short exons
(length less than the splice_penalty, approx) which may be
skipped. The intron penalties should not be set to less that the
maximum expected random match between the sequences (typically 10-15
bp) in order to avoid spurious matches.
The algorithm has the following steps:
<ol>
<li> A first-pass Smith-Waterman scan is done to locate the score, start
and end of the maximal scoring segment (including introns of
course). No other alignment information is retained.
<li> Subsequences corresponding to the maximal-scoring segments are
extracted. If the product of these subsequences' lengths is less than
the area parameter then the segments are re-aligned using the
Needleman-Wunsch algorithm, which in this instance will give the same
result as the Smith-Waterman since they are guaranteed to align
end-to-end.
<li> If the product of lengths exceeds the area threshold then the
alignment is recursively broken down by splitting the EST in half and
finding the genome position which aligns with the EST mid-point. The
problem then reduces to aligning the left-hand and right-hand portions
of the sequences separately and merging the result.
</ol>
The worst-case run-time for the algorithm is about 3 times as long as
would be taken to align using a quadratic-space program. In practice
the maximal-scoring segment is often much shorter than the full genome
length so the program runs only about 1.5 times slower.
<H2>
References
</H2>
<ol>
<li>Mott R. (1997) EST_GENOME: a program to align spliced DNA sequences to
unspliced genomic DNA. Comput. Applic. 13:477-478
<li>Huang X (1994) On global sequence alignment. Comput. Applic. Biosci.
10:227-235.
<li>Myers, EW and Miller, W (1988) Optimal alignments in linear space.
Comput. Applic. Biosci. 4:11-17
<li>Smith, TE and Waterman, MS (1981) Identification of common molecular
subsequences. J. Mol. Biol. 147:195-197
</ol>
<H2>
Warnings
</H2>
None.
<H2>
Diagnostic Error Messages
</H2>
None.
<H2>
Exit status
</H2>
It returns 0 unless an error occurs.
<H2>
Known bugs
</H2>
None.
<h2><a name="See also">See also</a></h2>
<table border cellpadding=4 bgcolor="#FFFFF0">
<tr><th>Program name</th><th>Description</th></tr>
<tr>
<td><a href="needle.html">needle</a></td>
<td>Needleman-Wunsch global alignment</td>
</tr>
<tr>
<td><a href="stretcher.html">stretcher</a></td>
<td>Finds the best global alignment between two sequences</td>
</tr>
</table>
<H2>
Author(s)
</H2>
This application was modified for inclusion in EMBOSS by
Peter Rice (pmr © ebi.ac.uk)
<br>
Informatics Division, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
<p>
The original program was est_genome, written by Richard Mott at the Sanger
Centre. The original version is available from <a
href="ftp://ftp.sanger.ac.uk/pub/pmr/est_genome.4.tar.Z">
ftp://ftp.sanger.ac.uk/pub/pmr/est_genome.4.tar.Z</a>
<H2>
History
</H2>
<H2>
Target users
</H2>
This program is intended to be used by everyone and everything, from naive users to embedded scripts.
<H2>
Comments
</H2>
<h3>Thu, 29 Mar 2001</h3>
I found est2genome having problems finding very short exons with the
default parameters.
<p>
With the folowing changes it detects also a 14bp exon correctly:
<p>
<pre>
mismatch 1 -> 3
intronpenalty 40 -> 20
splicepenalty 20 -> 10
minscore 30 -> 10
</pre>
<pre>
Dr. David Bauer
GenProfile AG, Max-Delbrueck-Center, Erwin-Negelein-Haus
Robert-Roessle-Str. 10, D-13125 Berlin, Germany
[email protected], Tel:49-30-94892165, FAX:49-30-94892151
</pre>
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