|
|
est2genome |
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.
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).
1. A first pass Smith-Waterman local alignment scan is done to find the start and end of the maximally scoring segments.
2. Subsequences corresponding to these segments are extracted
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.
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.
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).
5. Then the best-scoring orientation is realigned assuming reverse splicing (CT/AC consensus). The overall best alignment is reported.
% est2genome Align EST and genomic DNA sequences Spliced EST nucleotide sequence(s): tembl:h45989 Unspliced genomic nucleotide sequence: tembl:z69719 Output file [h45989.est2genome]: |
Go to the input files for this example
Go to the output files for this example
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
|
| Standard (Mandatory) qualifiers | Allowed values | Default | |||||||
|---|---|---|---|---|---|---|---|---|---|
| [-estsequence] (Parameter 1) |
Spliced EST nucleotide sequence(s) | Readable sequence(s) | Required | ||||||
| [-genomesequence] (Parameter 2) |
Unspliced genomic nucleotide sequence | Readable sequence | Required | ||||||
| [-outfile] (Parameter 3) |
Output file name | Output file | <*>.est2genome | ||||||
| Additional (Optional) qualifiers | Allowed values | Default | |||||||
| -match | Score for matching two bases | Any integer value | 1 | ||||||
| -mismatch | Cost for mismatching two bases | Any integer value | 1 | ||||||
| -gappenalty | Cost for deleting a single base in either sequence, excluding introns | Any integer value | 2 | ||||||
| -intronpenalty | Cost for an intron, independent of length. | Any integer value | 40 | ||||||
| -splicepenalty | Cost for an intron, independent of length and starting/ending on donor-acceptor sites | Any integer value | 20 | ||||||
| -minscore | Exclude alignments with scores below this threshold score. | Any integer value | 30 | ||||||
| Advanced (Unprompted) qualifiers | Allowed values | Default | |||||||
| -reverse | Reverse the orientation of the EST sequence | Boolean value Yes/No | No | ||||||
| -[no]splice | Use donor and acceptor splice sites. If you want to ignore donor-acceptor sites then set this to be false. | Boolean value Yes/No | Yes | ||||||
| -mode | 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. |
|
both | ||||||
| -[no]best | You can print out all comparisons instead of just the best one by setting this to be false. | Boolean value Yes/No | Yes | ||||||
| -space | 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 | 10.0 | ||||||
| -shuffle | Shuffle | Any integer value | 0 | ||||||
| -seed | Random number seed | Any integer value | 20825 | ||||||
| -align | 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). | Boolean value Yes/No | No | ||||||
| -width | Alignment width | Any integer value | 50 | ||||||
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: est@watson.wustl.edu
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 (info@image.llnl.gov) 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
//
|
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: vega@sanger.ac.uk Clone requests:
RL clonerequest@sanger.ac.uk
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: vega@sanger.ac.uk
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
[Part of this file has been deleted for brevity]
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
//
|
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. |
The score for Exon segments is the alignment score excluding flanking intron penalties. The Span score is the total including the intron costs.
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 +Intron (forward, splice sites GT/AG) or -Intron (reverse, splice sites CT/AC), or ?Intron (unknown direction). Segment entries give the alignment as a series of ungapped matching segments.
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.
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 L*gap_penaltyand the cost in the genome is
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)
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.
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:
| Program name | Description |
|---|---|
| needle | Needleman-Wunsch global alignment |
| stretcher | Finds the best global alignment between two sequences |
The original program was est_genome, written by Richard Mott at the Sanger Centre. The original version is available from ftp://ftp.sanger.ac.uk/pub/pmr/est_genome.4.tar.Z
With the folowing changes it detects also a 14bp exon correctly:
mismatch 1 -> 3 intronpenalty 40 -> 20 splicepenalty 20 -> 10 minscore 30 -> 10
Dr. David Bauer GenProfile AG, Max-Delbrueck-Center, Erwin-Negelein-Haus Robert-Roessle-Str. 10, D-13125 Berlin, Germany bauer@genprofile.com, Tel:49-30-94892165, FAX:49-30-94892151