|
est2genome |
Function
Align EST and genomic DNA sequencesDescription
est2genome 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.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).
Algorithm
The program uses a linear-space divide-and-conquer strategy (Myers and Miller, 1988; Huang, 1994) to limit memory use: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.
Usage
Here is a sample session with est2genome
% est2genome Align EST and genomic DNA sequences EST sequence(s): tembl:hs989235 Genomic sequence: tembl:hsnfg9 Output file [hs989235.est2genome]: |
Go to the input files for this example
Go to the output files for this example
Command line arguments
Standard (Mandatory) qualifiers:
[-estsequence] seqall EST sequence(s)
[-genomesequence] sequence Genomic sequence
[-outfile] outfile Output file name
Additional (Optional) qualifiers:
-match integer Score for matching two bases
-mismatch integer Cost for mismatching two bases
-gappenalty integer Cost for deleting a single base in either
sequence, excluding introns
-intronpenalty integer Cost for an intron, independent of length.
-splicepenalty integer Cost for an intron, independent of length
and starting/ending on donor-acceptor sites
-minscore integer Exclude alignments with scores below this
threshold score.
Advanced (Unprompted) qualifiers:
-reverse boolean Reverse the orientation of the EST sequence
-[no]splice boolean Use donor and acceptor splice sites. If you
want to ignore donor-acceptor sites then set
this to be false.
-mode menu 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.
-[no]best boolean You can print out all comparisons instead of
just the best one by setting this to be
false.
-space float 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)
-shuffle integer Shuffle
-seed integer Random number seed
-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 Alignment width
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 deaths
|
| Standard (Mandatory) qualifiers | Allowed values | Default | |||||||
|---|---|---|---|---|---|---|---|---|---|
| [-estsequence] (Parameter 1) |
EST sequence(s) | Readable sequence(s) | Required | ||||||
| [-genomesequence] (Parameter 2) |
Genomic sequence | Readable sequence | Required | ||||||
| [-outfile] (Parameter 3) |
Output file name | Output file | <sequence>.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 | ||||||
Input file format
est2genome reads two nucleotide sequences. The first is an EST sequence (a single read or a finished cDNA). The second is a genomic finished sequence.Input files for usage example
'tembl:hs989235' is a sequence entry in the example nucleic acid database 'tembl'
Database entry: tembl:hs989235
ID HS989235 standard; RNA; EST; 495 BP.
XX
AC H45989;
XX
SV H45989.1
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; Primates; Catarrhini; Hominidae; 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 RZPD; IMAGp998F03326; IMAGp998F03326.
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 /db_xref="taxon:9606"
FT /db_xref="ESTLIB:300"
FT /db_xref="RZPD:IMAGp998F03326"
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 /sex="Male"
FT /organism="Homo sapiens"
FT /clone="IMAGE:177794"
FT /clone_lib="Soares adult brain N2b5HB55Y"
FT /dev_stage="55-year old"
FT /lab_host="DH10B (ampicillin resistant)"
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
//
|
Database entry: tembl:hsnfg9
ID HSNFG9 standard; DNA; HUM; 33760 BP.
XX
AC Z69719;
XX
SV Z69719.1
XX
DT 26-FEB-1996 (Rel. 46, Created)
DT 22-NOV-1999 (Rel. 61, Last updated, Version 3)
XX
DE Human DNA sequence from cosmid NFG9 from a contig from the tip of the short
DE arm of chromosome 16, spanning 2Mb of 16p13.3. Contains Interleukin 9
DE Receptor Pseudogene, repeat polymorphism, ESTs, CpG islands and endogenous
DE retroviral DNA.
XX
KW 16p13.3; CpG island; Interleukin 9 Receptor Pseudogene;
KW repeat polymorphism.
XX
OS Homo sapiens (human)
OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
OC Eutheria; Primates; Catarrhini; Hominidae; Homo.
XX
RN [1]
RP 1-33760
RA Kershaw J.;
RT ;
RL Submitted (22-FEB-1996) to the EMBL/GenBank/DDBJ databases.
RL Sanger Centre, Hinxton, Cambridgeshire, CB10 1RQ, England. E-mail enquires:
RL humquery@sanger.ac.uk
XX
CC IMPORTANT: This sequence is not the entire insert of clone
CC NFG9. It may be shorter because we only sequence overlapping
CC sections once, or longer because we arrange for a small
CC overlap between neighbouring submissions.
XX
CC The true left end of clone NFG9 is at 1 in this sequence.
CC The true left end of clone RA36 is at 25872.
XX
CC NFG9 is from a 280kb clone contig extending from the telomere of 16p.
CC Higgs D.R., Flint J. unpublished. MRC Molecular Haematology Unit,
CC Institute of Molecular Medicine, Oxford.
CC NFG9 is from the library CV007K. Choo et al.,(1986) Gene 46. 277-286.
XX
FH Key Location/Qualifiers
FH
FT source 1..33760
FT /chromosome="16"
FT /db_xref="taxon:9606"
FT /organism="Homo sapiens"
FT /map="16p13.3"
FT /clone_lib="CV007K"
[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
//
|
Output file format
Output files for usage example
File: hs989235.est2genome
Note Best alignment is between forward est and forward genome, but splice sites imply REVERSED GENE Exon 163 91.8 25685 25874 HSNFG9 1 193 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. -Intron -20 0.0 25875 26278 HSNFG9 Exon 207 98.1 26279 26492 HSNFG9 194 407 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. -Intron -20 0.0 26493 27390 HSNFG9 Exon 63 86.4 27391 27476 HSNFG9 408 494 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Span 393 93.6 25685 27476 HSNFG9 1 494 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 14 83.3 25685 25702 HSNFG9 1 18 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 28 85.7 25703 25737 HSNFG9 20 54 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 4 100.0 25738 25741 HSNFG9 56 59 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 13 100.0 25742 25754 HSNFG9 61 73 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 4 100.0 25756 25759 HSNFG9 74 77 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 110 97.4 25760 25874 HSNFG9 79 193 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 37 100.0 26279 26315 HSNFG9 194 230 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 162 98.8 26317 26480 HSNFG9 231 394 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 12 100.0 26481 26492 HSNFG9 396 407 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 16 100.0 27391 27406 HSNFG9 408 423 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 10 91.7 27407 27418 HSNFG9 425 436 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 19 95.2 27419 27439 HSNFG9 438 458 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. Segment 24 80.6 27441 27476 HSNFG9 459 494 HS989235 yo13c02.s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA sequence. |
MSP type segments
There are four types of segment,- each gapped Exon
- each Intron (marked with a ? if it does not start GT and end AG)
- the complete alignment Span
- individual ungapped matching Segments.
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.
Full alignment
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)Data files
NoneNotes
est2genome uses a linear-space dynamic-programming algorithm. It has the following parameters:
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:
- 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.
- 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.
- 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.
References
- Mott R. (1997) EST_GENOME: a program to align spliced DNA sequences to unspliced genomic DNA. Comput. Applic. 13:477-478
- Huang X (1994) On global sequence alignment. Comput. Applic. Biosci. 10:227-235.
- Myers, EW and Miller, W (1988) Optimal alignments in linear space. Comput. Applic. Biosci. 4:11-17
- Smith, TE and Waterman, MS (1981) Identification of common molecular subseqeunces. J. Mol. Biol. 147:195-197
Warnings
None.Diagnostic Error Messages
None.Exit status
It return 0 unless an error occurs.Known bugs
None.See also
| Program name | Description |
|---|---|
| needle | Needleman-Wunsch global alignment |
| stretcher | Finds the best global alignment between two sequences |
Author(s)
This application was modified for inclusion in EMBOSS by Peter Rice (pmr © ebi.ac.uk)Informatics Division, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
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
History
Target users
This program is intended to be used by everyone and everything, from naive users to embedded scripts.Comments
Thu, 29 Mar 2001
I found est2genome having problems finding very short exons with the default parameters.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