
                                est2genome 



Function

   Align EST and genomic DNA sequences

Description

   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). H2> 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    (Both strands)
   forward (Forward strand only)
   reverse (Reverse strand only)
   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 17-1
8
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, basa
l
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        6
0
     aacctgagcg ggactggnag aaggagcaga gggaggcagc acccggcgtg acggnagtgt       12
0
     gtggggcact caggccttcc gcagtgtcat ctgccacacg gaaggcacgg ccacgggcag       18
0
     gggggtctat gatcttctgc atgcccagct ggcatggccc cacgtagagt ggnntggcgt       24
0
     ctcggtgctg gtcagcgaca cgttgtcctg gctgggcagg tccagctccc ggaggacctg       30
0
     gggcttcagc ttcccgtagc gctggctgca gtgacggatg ctcttgcgct gccatttctg       36
0
     ggtgctgtca ctgtccttgc tcactccaaa ccagttcggc ggtccccctg cggatggtct       42
0
     gtgttgatgg acgtttgggc tttgcagcac cggccgccga gttcatggtn gggtnaagag       48
0
     atttgggttt tttcn                                                        49
5
//

  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 shor
t
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     3084
0
     tctcctgggg ttcccacctc cacaaaaacc cccactgcta ggagtgcagg caggagggga     3090
0
     cctgagaacc gacagttata ggtcctgcgg gtgggcagtg ctgggtgttc tggtctgccc     3096
0
     cacccctgtg tgcctagatc cccatctggg cctcaagtgg gtgggattcc aaaggaagag     3102
0
     ccggagtagg cgtggggagg ggcaggccca ggctggacaa agagtctggc cagggagcgg     3108
0
     cacattgccc tcccagagac agtggctcag tgtccaggcc ttccccaggc gcacagtggg     3114
0
     ctcttgttcc cagaaagccc ctcgggggga tccaaacagt gtctccccca ccccgctgac     3120
0
     ccctcagtgt atggggaaac cgtggcccac ggaaggcctc actgcctggg gtcacacagc     3126
0
     atctgagtca ctgcagcagc ctcacagctg ccagcccagg cccagcccca tcaggagaca     3132
0
     cccaaagcca cagtgcatcc caggaccagc tgggggggct gcgggcagga ctctcgatga     3138
0
     ggctgaggga cgaggagggt caagggagcc actggcgcca tgcatgctga cgtcccctct     3144
0
     ggctgcctgc agagcctggt gtggaagggc tgagtggggg atggtggaga gtcctgttaa     3150
0
     ctcaggtttc tgctctgggg atgtctgggc acccatcaag ctggccgcgt gcacaggtgc     3156
0
     agggagagcc agaaagcagg agccgatgca gggaggccac tggggacagc ccaggctgat     3162
0
     gcttgggccc catgtgtctc caccacctac aaccctaagc aagcctcagc tttcccatct     3168
0
     ggaaatcagg ggtcacagca gtgcctggca cagtagcagc ggctgactcc atcacagggt     3174
0
     ggtgtagcct gtgggtactt ggcactctct gaggggcagg agctgggggg tgaaaggacc     3180
0
     ctagagcata tgcaacaaga gggcagccct ggggacacct ggggacagaa ccctccaaag     3186
0
     gtgtcgagtt tgggaagaga ctagagagaa gctctggcca gtccaggcat agacagtggc     3192
0
     cacagccagt ggagagctgc atcctcaggt gtgagcagca accacctctg tactcaggcc     3198
0
     tgccctgcac actcacagga ccatgctggc agggacaact ggcggcggag ttgactgcca     3204
0
     accccggggc cagaaccatc aagcctgggc tctgctccgc ccaaggaact gcctgctgcc     3210
0
     gaggtcagct ggagcaaggg gcctcacccc gggacacctt cccagacgtg tcctcagctc     3216
0
     acatgagcct catcccaggg ggatgtggct cctccagcat ccccacccac acgctgctct     3222
0
     ctgaccctca gtcttctgtt tgactcctaa tctgaagctc aatcctagat ctcccttgag     3228
0
     aagggggtca ccagctgtct ggcagcccag cctccaggtc ttctggatta atgaagggaa     3234
0
     agtcacctgg cctctctgcc ttgtctatta atggcatcat gctgagaatg atatttgcta     3240
0
     ggccctttgc aaaccccaaa gtgctcttca accctcccag tgaagcctct tcttttctgt     3246
0
     ggaagaaatg aggttcaggg tggagcaggg caggcctgag acctttgcag ggttctctcc     3252
0
     aggtccccag caggacagac tggcaccctg cctcccctca tcaccctaga caaggagaca     3258
0
     gaacaagagg ttccctgcta caggccatct gtgagggaag ccgccctagg gcctgtagac     3264
0
     acaggaatcc ctgaggacct gacctgtgag ggtagtgcac aaaggggcca gcacttggca     3270
0
     ggaggggggg gggcactgcc ccaaggctca gctagcaaat gtggcacagg ggtcaccaga     3276
0
     gctaaacccc tgactcagtt gggtctgaca ggggctgaca tggcagacac acccaggaat     3282
0
     caggggacac caagtgcagc tcagggcacc tgtccaggcc acacagtcag aaaggggatg     3288
0
     gcagcaagga cttagctaca ctagattctg ggggtaaact gcctggtatg ctggtcactg     3294
0
     ctagtcccca gtctggagtc tagctgggtc tcaggagtta ggcgaaaaca ccctccccag     3300
0
     gctgcaggtg ggagaggccc acatcccctg cacacgtctg gccagaggac agatgggcag     3306
0
     cccagtcacc agtcagagcc ctccagaggt gtccctgact gaccctacac acatgcaccc     3312
0
     aggtgcccag gcacccttgg gctcagcaac cctgcaaccc cctcccagga cccaccagaa     3318
0
     gcaggatagg actagagagg ccacaggagg gaaaccaagt cagagcagaa atggcttcgg     3324
0
     tcctcagcag cctggctcag cttcctcaaa ccagatcctg actgatcaca ctggtctgtc     3330
0
     taacccctgg gaggggtcct ctgtatccat cttacagata aggaaactga ggctcagaga     3336
0
     agcccatcac tgcctaaggt cccagggcct ataagggagc tcaaagcctt gggccaggtc     3342
0
     tgcccaggag ctgcagtgga agggaccctg tctgcagacc cccagaagac aaggcagacc     3348
0
     acctgggttc ttcagccttg tggctgtgga cggctgtcag acccttctaa gaccccttgc     3354
0
     cacctgctcc atcaggggca tctcagttga agaaggaagg actcaccccc aaaatcgtcc     3360
0
     aactcagaaa aaaaggcaga agccaaggaa tccaatcact gggcaaaatg tgatcctggc     3366
0
     acagacactg aggtggggga actggagccg gtgtggcgga ggccctcaca gccaagagca     3372
0
     actgggggtg ccctgggcag ggactgtagc tgggaagatc                           3376
0
//

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,
    1. each gapped Exon
    2. each Intron (marked with a ? if it does not start GT and end AG)
    3. the complete alignment Span
    4. 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

   None

Notes

   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_penalty

   and 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 A
G
                                     (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:
    1. 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.
    2. 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.
    3. 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.

   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.

References

    1. Mott R. (1997) EST_GENOME: a program to align spliced DNA
       sequences to unspliced genomic DNA. Comput. Applic. 13:477-478
    2. Huang X (1994) On global sequence alignment. Comput. Applic.
       Biosci. 10:227-235.
    3. Myers, EW and Miller, W (1988) Optimal alignments in linear space.
       Comput. Applic. Biosci. 4:11-17
    4. 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
