est2genome Wiki The master copies of EMBOSS documentation are available at http://emboss.open-bio.org/wiki/Appdocs on the EMBOSS Wiki. Please help by correcting and extending the Wiki pages. Function Align EST sequences to genomic DNA sequence Description est2genome aids the prediction of genes by sequence homology. It aligns a set of spliced nucleotide sequences (ESTs cDNAs or mRNAs) to an unspliced genomic DNA sequence, inserting introns of arbitrary length when needed. Where feasible introns start and stop at the splice consensus dinucleotides GT and AG. By default, est2genome makes three alignments: First it compares both strands of the spliced sequence against the forward strand of the genomic sequence, 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). By default, only the overall maximum-scoring alignment is reported, and then if it scores higher than a specific minimum threshold score. Optionally, all comparisons may be 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. 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, end and score of the maximally scoring segments (including introns of course). No other alignment information is retained. 2. Subsequences corresponding to these segments are extracted 3a. If the product of the subsequences' lengths is less than a user-defined threshold (-space parameter), 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 EST mid-point. The process is repeated until the product of the lengths is less than the threshold. The problem reduces to aligning the left-hand and right-hand portions of the sequences separately and merging the result. 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. 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. 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. Usage Here is a sample session with est2genome % est2genome Align EST sequences to genomic DNA sequence 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 Command line arguments Align EST sequences to genomic DNA sequence Version: EMBOSS:6.4.0.0 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]usesplice 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 comparison 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 comparison 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 first file to standard output -filter boolean Read first file from standard input, write first file to 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 and exit. 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 -version boolean Report version number and exit 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:h45989' is a sequence entry in the example nucleic acid database 'tembl' Database entry: tembl:h45989 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 ImaGenes; ENSEp780A0214D. DR ImaGenes; ENSEp780A044Q. DR ImaGenes; HU3_p972A0639D. DR ImaGenes; HU3_p972B1110Q. DR ImaGenes; HU3_p983A0639D. DR ImaGenes; HU4_p940A0622D. DR ImaGenes; IMAGp956A0431Q. DR ImaGenes; IMAGp998F03326Q. DR ImaGenes; RZPDp1096A101D. DR ImaGenes; RZPDp1096A191Q. DR ImaGenes; RZPDp200A0214D. 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="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 // Database entry: tembl:z69719 ID Z69719; SV 1; linear; genomic DNA; STD; HUM; 33760 BP. XX AC Z69719; XX DT 26-FEB-1996 (Rel. 46, Created) DT 13-JAN-2009 (Rel. 99, Last updated, Version 7) 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 (09-JAN-2009) 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: Geneservice RL (http://www.geneservice.co.uk/) and BACPAC Resources RL (http://bacpac.chori.org/) XX DR EMBL-CON; GL000124. DR GDB; 11502921. DR RFAM; RF00017; SRP_euk_arch. 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 [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: h45989.est2genome 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 se quence. -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 se quence. -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 se quence. Span 393 93.6 25685 27476 Z69719 1 494 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 14 83.3 25685 25702 Z69719 1 18 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 28 85.7 25703 25737 Z69719 20 54 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 4 100.0 25738 25741 Z69719 56 59 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 13 100.0 25742 25754 Z69719 61 73 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 4 100.0 25756 25759 Z69719 74 77 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 110 97.4 25760 25874 Z69719 79 193 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 37 100.0 26279 26315 Z69719 194 230 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 162 98.8 26317 26480 Z69719 231 394 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 12 100.0 26481 26492 Z69719 396 407 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 16 100.0 27391 27406 Z69719 408 423 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 10 91.7 27407 27418 Z69719 425 436 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 19 95.2 27419 27439 Z69719 438 458 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. Segment 24 80.6 27441 27476 Z69719 459 494 H45989 yo13c02. s1 Soares adult brain N2b5HB55Y Homo sapiens cDNA clone IMAGE:177794 3', mRNA se quence. 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 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. 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 subsequences. J. Mol. Biol. 147:195-197 Warnings None. Diagnostic Error Messages None. Exit status It returns 0 unless an error occurs. Known bugs None. See also Program name Description needle Needleman-Wunsch global alignment of two sequences needleall Many-to-many pairwise alignments of two sequence sets stretcher Needleman-Wunsch rapid global alignment of two sequences Author(s) This application was modified for inclusion in EMBOSS by Peter Rice European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK Please report all bugs to the EMBOSS bug team (emboss-bug (c) emboss.open-bio.org) not to the original author. 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