Difference between revisions of "Texas Xenopus Genome Project/Species Identification"
From Marcotte Lab
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− | == | + | == Designed == |
− | * | + | === XENTR_CHORI-219_ID.2 (hoxa5) === |
− | ** | + | * Only cut XENTR - [http://rebase.neb.com/rebase/enz/PsrI.html PsrI](119, 151). |
− | ** | + | * Only cut XENLA - None. |
+ | * PCR primer (designed by Primer3) | ||
+ | <pre> | ||
+ | OLIGO start len tm gc% any 3' seq | ||
+ | LEFT PRIMER 24 20 59.99 45.00 2.00 0.00 caagagccacaaatcaagca | ||
+ | RIGHT PRIMER 221 20 59.92 50.00 5.00 1.00 agatccatgccattgtagcc | ||
+ | </pre> | ||
+ | |||
+ | <pre> | ||
+ | XENLA_CH219-206K 1 + gtgctatagacgcgcaaaacgaccaagagccacaaatcaagcacacatatcaaaaaacaaatga 64 | ||
+ | XENTR_NM_0010114 1 + gtgctatagacgcgcaaaacgaccaagagccacaaatcaagcacacatatcaaaaaacaaatga 64 | ||
+ | **************************************************************** | ||
+ | >>>>>>>>>>>>>>>>>>>> | ||
+ | XENLA_CH219-206K 65 + gctcttattttgtaaactcattttgcggtcgctatccaaatggcccggactaccagttacataa 128 | ||
+ | XENTR_NM_0010114 65 + gctcttattttgtaaactcattttgcggtcgctatccaaatggcccggactaccagttacataa 128 | ||
+ | **************************************************************** | ||
+ | |||
+ | XENLA_CH219-206K 129 + ttatggagatcagagctcggtgagcgagcaatacagggatacaacgagcatgcattccagcagg 192 | ||
+ | XENTR_NM_0010114 129 + ttatggagatcacagctcggtgaacgagcaatacagggatacaacgagcatgcattccagcagg 192 | ||
+ | ************ ********** **************************************** | ||
+ | |||
+ | XENLA_CH219-206K 193 + tacggatacggctacaatggcatggatctcagcgttgggcgctcagcttccaaccactttagtg 256 | ||
+ | XENTR_NM_0010114 193 + tacggatacggctacaatggcatggatctcagcgttgggcgctcagcttccaaccactttagtg 256 | ||
+ | **************************************************************** | ||
+ | <<<<<<<<<<<<<<<<<<<< | ||
+ | XENLA_CH219-206K 257 + ccaatgacagagcaaggaattatccatccaatccgagctcctctacagagccaaggtataacca 320 | ||
+ | XENTR_NM_0010114 257 + ccaatgacagagcaaggaattatccatccaatccgagctcctctacagagccaaggtataacca 320 | ||
+ | **************************************************************** | ||
+ | |||
+ | XENLA_CH219-206K 321 + acctgcagcctct 333(1) | ||
+ | XENTR_NM_0010114 321 + acctgcagcgtct 333(1) | ||
+ | ********* *** | ||
+ | </pre> | ||
+ | |||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.2.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.2.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.25 (hoxa3) === | ||
+ | * Only cut XENTR - BccI(136), BfiI(110), BglII(21), BstEII(140), Eco57I(72), Eco57MI(72), MboII(78), MmeI(115), PflMI(134), PleI(118), Tsp45I(99), TspDTI(59), TspRI(123,178) | ||
+ | * Only cut XENLA - BciVi(136,167), BspMI(156), EcoP15I(193), HphI(136), MslI(105), NspI(157), SacI(44,122), SphI(157), TspGWI(157) | ||
+ | * PCR primer | ||
+ | <pre> | ||
+ | OLIGO start len tm gc% any 3' seq | ||
+ | LEFT PRIMER 161 20 60.21 50.00 7.00 2.00 cctcaccgagaggcaaatta | ||
+ | RIGHT PRIMER 351 20 59.68 60.00 5.00 3.00 gaggctcataggggacactg | ||
+ | </pre> | ||
+ | |||
+ | <pre> | ||
+ | XENLA_CH219-206K 1 + gtgagagctgtgcaggagacaaaagtcccccggggcaatcctcttccaaaagggcccgcactgc 64 | ||
+ | XENTR_NM_0011274 1 + gtgagagttgtgctggagacaaaagccccccggggcaatcctcttccaagagggcccgcactgc 64 | ||
+ | ******* ***** *********** *********************** ************** | ||
+ | |||
+ | XENLA_CH219-206K 65 + ttacacaagcgctcagctggtagaactggaaaaagagttccactttaacagatacctgtgcaga 128 | ||
+ | XENTR_NM_0011274 65 + ttacacaagcgctcagctggtagaactggaaaaagagttccactttaacagatacctgtgcaga 128 | ||
+ | **************************************************************** | ||
+ | |||
+ | XENLA_CH219-206K 129 + cccaggagggtggagatggccaatctactcaacctcaccgagaggcaaattaagatctggtttc 192 | ||
+ | XENTR_NM_0011274 129 + cccaggagggtggagatggccaatctgctcaacctcaccgagaggcaaattaagatctggtttc 192 | ||
+ | ************************** ************************************* | ||
+ | >>>>>>>>>>>>>>>>>>>> | ||
+ | XENLA_CH219-206K 193 + agaacaggcgaatgaaatacaaaaaggatcaaaaagggaaatccatgatgacctcttcaggagg 256 | ||
+ | XENTR_NM_0011274 193 + agaacaggcgaatgaaatacaaaaaggatcaaaaagggaaatccatgatgacctcttcaggagg 256 | ||
+ | **************************************************************** | ||
+ | |||
+ | XENLA_CH219-206K 257 + acagtcaccatgtaggagcccggtgccagctccatctgttggaggttacctaaactctatgcat 320 | ||
+ | XENTR_NM_0011274 257 + gcagtcaccatgtaggagcccagtgccgactccatctgttggaggttacctaaactctatgcat 320 | ||
+ | ******************** ***** *********************************** | ||
+ | |||
+ | XENLA_CH219-206K 321 + tctttggtaaacagtgtcccctatgagcctcagtctccccctg 363(1) | ||
+ | XENTR_NM_0011274 321 + tctttggtaaacagtgtcccctatgagcctcagtctcccccag 363(1) | ||
+ | ***************************************** * | ||
+ | <<<<<<<<<<<<<<<<<<<< | ||
+ | </pre> | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.25.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.25.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.28 (pitx-1) === | ||
+ | * Only cut XENTR - SfaNI(46),TauI(52) | ||
+ | * Only cut XENLA - BciVI(78),FauI(42),Hpy99I(72) | ||
+ | * PCR primer | ||
+ | <pre> | ||
+ | OLIGO start len tm gc% any 3' seq | ||
+ | LEFT PRIMER 42 20 60.12 55.00 5.00 2.00 gaaccagcagatggacctgt | ||
+ | RIGHT PRIMER 194 20 59.84 50.00 4.00 0.00 aaggtgaagctcttggtgga | ||
+ | </pre> | ||
+ | <pre> | ||
+ | XENLA_CH219-166K 1 + tggttcaagaaccgccgagccaagtggaggaagagggagcggaaccagcagatggacctgtgta 64 | ||
+ | XENTR_NM_0010074 1 + tggttcaagaaccgcagagccaagtggaggaagagggagcggaaccagcagatggacctgtgca 64 | ||
+ | *************** ********************************************** * | ||
+ | >>>>>>>>>>>>>>>>>>> | ||
+ | XENLA_CH219-166K 65 + agaatggttacgtgccccagttcagcgggctcatgcagccgtacgacgagatgtacgcaggata 128 | ||
+ | XENTR_NM_0010074 65 + agaatggctacgtgccccagttcagcggcctgatgcagccctacgatgagatgtacgctggcta 128 | ||
+ | ******* ******************** ** ******** ***** *********** ** ** | ||
+ | |||
+ | XENLA_CH219-166K 129 + cccctacaacaactgggccacaaaaagcctcacccctgcccccctgtccaccaagagcttcacc 192 | ||
+ | XENTR_NM_0010074 129 + cccgtacaacaactgggccaccaaaagcctcacccctgcccccctgtccaccaagagcttcacc 192 | ||
+ | *** ***************** ****************************************** | ||
+ | <<<<<<<<<<<<<<<<<< | ||
+ | XENLA_CH219-166K 193 + ttcttcaactccatgagtcccttgtcttcccagtccatgttctccggccccagctccatctctt 256 | ||
+ | XENTR_NM_0010074 193 + ttcttcaactccatgagtccgctgtcctcccagtccatgttctctggccccagctccatctctt 256 | ||
+ | ******************** **** ***************** ******************* | ||
+ | << | ||
+ | XENLA_CH219-166K 257 + ccatgagcatgccctccagcatgggtcactctgcggtgccaggcatggccaactc 311(1) | ||
+ | XENTR_NM_0010074 257 + ccatgagcatgccctccagcatgggccactcggcggtgcccggcatgcccaactc 311(1) | ||
+ | ************************* ***** ******** ****** ******* | ||
+ | </pre> | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.28.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.28.muscle|MUSCLE file]] | ||
+ | |||
+ | == Candidates == | ||
+ | === XENTR_CHORI-219_ID.3 (hoxa5) === | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.3.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.3.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.24 (hoxa3) === | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.24.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.24.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.26 (hoxa3) === | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.26.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.26.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.27 (pitx-1) === | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.27.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.27.muscle|MUSCLE file]] | ||
+ | |||
+ | === XENTR_CHORI-219_ID.29 (pitx-1) === | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.29.fasta|FASTA file]] | ||
+ | * [[:xdata:ID/XENTR_CHORI-219_ID.29.muscle|MUSCLE file]] | ||
== Selection procedure == | == Selection procedure == | ||
Line 28: | Line 156: | ||
*#* NM_001142220 phosphoribosylformylglycinamidine synthase (pfas) [http://www.ncbi.nlm.nih.gov/nuccore/213983084 NCBI][http://www.xenbase.org/gene/showgene.do?method=displayGeneSummary&geneId=6001437 XenBase] | *#* NM_001142220 phosphoribosylformylglycinamidine synthase (pfas) [http://www.ncbi.nlm.nih.gov/nuccore/213983084 NCBI][http://www.xenbase.org/gene/showgene.do?method=displayGeneSummary&geneId=6001437 XenBase] | ||
+ | * Run MUSCLE (version 4.0, with default option) for multiple sequence alignment. I found that most of candidate sequences have duplications. So I filtered them out based on 'Self' mapping information from MUSCLE output (if a sequence is mapped itself longer than 50 bp). Finally 10 fragments are selected. | ||
+ | ** hoxa3 - XENTR_CHORI-219_ID.24.muscle, XENTR_CHORI-219_ID.25.muscle, XENTR_CHORI-219_ID.26.muscle | ||
+ | ** pitx-1 - XENTR_CHORI-219_ID.27.muscle, XENTR_CHORI-219_ID.28.muscle, XENTR_CHORI-219_ID.29.muscle | ||
+ | ** hoxa5 - XENTR_CHORI-219_ID.2.muscle, XENTR_CHORI-219_ID.3.muscle | ||
+ | :<pre> $ mus4 -i XENTR_CHORI.fasta -o XENTR_CHORI.muscle </pre> | ||
− | + | Here's the example of Self duplication. | |
− | + | ||
− | + | ||
− | + | ||
<pre> | <pre> | ||
− | + | >XENTR_NM_001113032_18 gi|163915026|ref|NM_001113032| | |
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− | + | 3 + ccaggggtacccagggcacaaataagcactcaccccaaatccccccctaactggccttcaggct 66 | |
− | + | ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||| | |
− | + | 138 + ccaggggtacccagggcacaaataagcactcaccccaaatctccccctaactggccttcaggct 201 | |
− | + | 67 + gggcccccttagcccataacaaggttacagatagttagaaacattggg 114 | |
− | + | ||||||||||||||||||||||||||||||||| ||||||||||||| | |
− | + | 202 + gggcccccttagcccataacaaggttacagatatatagaaacattggg 249 | |
</pre> | </pre> | ||
− | |||
---- | ---- | ||
[[Category:XenopusGenome]] | [[Category:XenopusGenome]] |
Latest revision as of 16:43, 9 December 2009
Contents |
Designed
XENTR_CHORI-219_ID.2 (hoxa5)
- Only cut XENTR - PsrI(119, 151).
- Only cut XENLA - None.
- PCR primer (designed by Primer3)
OLIGO start len tm gc% any 3' seq LEFT PRIMER 24 20 59.99 45.00 2.00 0.00 caagagccacaaatcaagca RIGHT PRIMER 221 20 59.92 50.00 5.00 1.00 agatccatgccattgtagcc
XENLA_CH219-206K 1 + gtgctatagacgcgcaaaacgaccaagagccacaaatcaagcacacatatcaaaaaacaaatga 64 XENTR_NM_0010114 1 + gtgctatagacgcgcaaaacgaccaagagccacaaatcaagcacacatatcaaaaaacaaatga 64 **************************************************************** >>>>>>>>>>>>>>>>>>>> XENLA_CH219-206K 65 + gctcttattttgtaaactcattttgcggtcgctatccaaatggcccggactaccagttacataa 128 XENTR_NM_0010114 65 + gctcttattttgtaaactcattttgcggtcgctatccaaatggcccggactaccagttacataa 128 **************************************************************** XENLA_CH219-206K 129 + ttatggagatcagagctcggtgagcgagcaatacagggatacaacgagcatgcattccagcagg 192 XENTR_NM_0010114 129 + ttatggagatcacagctcggtgaacgagcaatacagggatacaacgagcatgcattccagcagg 192 ************ ********** **************************************** XENLA_CH219-206K 193 + tacggatacggctacaatggcatggatctcagcgttgggcgctcagcttccaaccactttagtg 256 XENTR_NM_0010114 193 + tacggatacggctacaatggcatggatctcagcgttgggcgctcagcttccaaccactttagtg 256 **************************************************************** <<<<<<<<<<<<<<<<<<<< XENLA_CH219-206K 257 + ccaatgacagagcaaggaattatccatccaatccgagctcctctacagagccaaggtataacca 320 XENTR_NM_0010114 257 + ccaatgacagagcaaggaattatccatccaatccgagctcctctacagagccaaggtataacca 320 **************************************************************** XENLA_CH219-206K 321 + acctgcagcctct 333(1) XENTR_NM_0010114 321 + acctgcagcgtct 333(1) ********* ***
XENTR_CHORI-219_ID.25 (hoxa3)
- Only cut XENTR - BccI(136), BfiI(110), BglII(21), BstEII(140), Eco57I(72), Eco57MI(72), MboII(78), MmeI(115), PflMI(134), PleI(118), Tsp45I(99), TspDTI(59), TspRI(123,178)
- Only cut XENLA - BciVi(136,167), BspMI(156), EcoP15I(193), HphI(136), MslI(105), NspI(157), SacI(44,122), SphI(157), TspGWI(157)
- PCR primer
OLIGO start len tm gc% any 3' seq LEFT PRIMER 161 20 60.21 50.00 7.00 2.00 cctcaccgagaggcaaatta RIGHT PRIMER 351 20 59.68 60.00 5.00 3.00 gaggctcataggggacactg
XENLA_CH219-206K 1 + gtgagagctgtgcaggagacaaaagtcccccggggcaatcctcttccaaaagggcccgcactgc 64 XENTR_NM_0011274 1 + gtgagagttgtgctggagacaaaagccccccggggcaatcctcttccaagagggcccgcactgc 64 ******* ***** *********** *********************** ************** XENLA_CH219-206K 65 + ttacacaagcgctcagctggtagaactggaaaaagagttccactttaacagatacctgtgcaga 128 XENTR_NM_0011274 65 + ttacacaagcgctcagctggtagaactggaaaaagagttccactttaacagatacctgtgcaga 128 **************************************************************** XENLA_CH219-206K 129 + cccaggagggtggagatggccaatctactcaacctcaccgagaggcaaattaagatctggtttc 192 XENTR_NM_0011274 129 + cccaggagggtggagatggccaatctgctcaacctcaccgagaggcaaattaagatctggtttc 192 ************************** ************************************* >>>>>>>>>>>>>>>>>>>> XENLA_CH219-206K 193 + agaacaggcgaatgaaatacaaaaaggatcaaaaagggaaatccatgatgacctcttcaggagg 256 XENTR_NM_0011274 193 + agaacaggcgaatgaaatacaaaaaggatcaaaaagggaaatccatgatgacctcttcaggagg 256 **************************************************************** XENLA_CH219-206K 257 + acagtcaccatgtaggagcccggtgccagctccatctgttggaggttacctaaactctatgcat 320 XENTR_NM_0011274 257 + gcagtcaccatgtaggagcccagtgccgactccatctgttggaggttacctaaactctatgcat 320 ******************** ***** *********************************** XENLA_CH219-206K 321 + tctttggtaaacagtgtcccctatgagcctcagtctccccctg 363(1) XENTR_NM_0011274 321 + tctttggtaaacagtgtcccctatgagcctcagtctcccccag 363(1) ***************************************** * <<<<<<<<<<<<<<<<<<<<
XENTR_CHORI-219_ID.28 (pitx-1)
- Only cut XENTR - SfaNI(46),TauI(52)
- Only cut XENLA - BciVI(78),FauI(42),Hpy99I(72)
- PCR primer
OLIGO start len tm gc% any 3' seq LEFT PRIMER 42 20 60.12 55.00 5.00 2.00 gaaccagcagatggacctgt RIGHT PRIMER 194 20 59.84 50.00 4.00 0.00 aaggtgaagctcttggtgga
XENLA_CH219-166K 1 + tggttcaagaaccgccgagccaagtggaggaagagggagcggaaccagcagatggacctgtgta 64 XENTR_NM_0010074 1 + tggttcaagaaccgcagagccaagtggaggaagagggagcggaaccagcagatggacctgtgca 64 *************** ********************************************** * >>>>>>>>>>>>>>>>>>> XENLA_CH219-166K 65 + agaatggttacgtgccccagttcagcgggctcatgcagccgtacgacgagatgtacgcaggata 128 XENTR_NM_0010074 65 + agaatggctacgtgccccagttcagcggcctgatgcagccctacgatgagatgtacgctggcta 128 ******* ******************** ** ******** ***** *********** ** ** XENLA_CH219-166K 129 + cccctacaacaactgggccacaaaaagcctcacccctgcccccctgtccaccaagagcttcacc 192 XENTR_NM_0010074 129 + cccgtacaacaactgggccaccaaaagcctcacccctgcccccctgtccaccaagagcttcacc 192 *** ***************** ****************************************** <<<<<<<<<<<<<<<<<< XENLA_CH219-166K 193 + ttcttcaactccatgagtcccttgtcttcccagtccatgttctccggccccagctccatctctt 256 XENTR_NM_0010074 193 + ttcttcaactccatgagtccgctgtcctcccagtccatgttctctggccccagctccatctctt 256 ******************** **** ***************** ******************* << XENLA_CH219-166K 257 + ccatgagcatgccctccagcatgggtcactctgcggtgccaggcatggccaactc 311(1) XENTR_NM_0010074 257 + ccatgagcatgccctccagcatgggccactcggcggtgcccggcatgcccaactc 311(1) ************************* ***** ******** ****** *******
Candidates
XENTR_CHORI-219_ID.3 (hoxa5)
XENTR_CHORI-219_ID.24 (hoxa3)
XENTR_CHORI-219_ID.26 (hoxa3)
XENTR_CHORI-219_ID.27 (pitx-1)
XENTR_CHORI-219_ID.29 (pitx-1)
Selection procedure
- Download X. tropicalis mRNA sequences from XenBase (Nov. 27, 2009 version).
- xdata:ID/XENTR_mRNA.xenbase20091127.fasta.gz 17 MB, gzipped.
- Download CHORI-219 sequences (from NCBI GenBank).
- xdata:ID/XENLA_CH219.fasta.gz 6.5 MB, gzipped. (CHORI-219 sequences. 29 BAC sequences from X. laeves genome)
- Run BLAT (version 3.4, with default option) to known CHORI BAC sequences.
- xdata:ID/XENTR_mRNA.XENLA_CH219.blat_pslx.gz 1.2 MB, gzipped.
blat XENLA_CH219.fasta XENTR_mRNA.xenbase20091127.fasta XENTR_mRNA.XENLA_CH219.blat_pslx -out=pslx
- Parse two BLAT output files with the following criteria.
- From X. tropicalis mRNA, only RefSeq (starts sith 'NM_') sequences are considered.
- Select X. tropicalis mRNA sequences which hit both CHORI-219 (minimum match length is 200 bp to be called as a 'hit'). I only consider 10 CHORI-219 BACs which we already knew that they are available ('74I8','204L9','197E3','71P23','36I4','35I18','262A22','20I13','206K7','166K18').
- Survey each hit blocks. If the hit block is less than 200 bp, discard it. 42 hit blocks from 8 mRNAs are selected.
- NM_001004837 Unnamed, predicted gene MGC69309 NCBIXenBase
- NM_001007499 paired-like homeodomain 1 (pitx-1) NCBIXenBase
- NM_001011405 Homeobox A5 (hoxa5) NCBIXebBase
- NM_001035121 CCAAT/enhancer binding protein (C/EBP), beta (cebpb) NCBIXenBase
- NM_001113032 LY6/PLAUR domain containing 6 (lypd6) NCBIXenBase
- NM_001127429 homeobox A3 (hoxa3) NCBIXenBase
- NM_001129937 SRY (sex determining region Y)-box 18 (sox18) NCBI XenBase
- NM_001142220 phosphoribosylformylglycinamidine synthase (pfas) NCBIXenBase
- Run MUSCLE (version 4.0, with default option) for multiple sequence alignment. I found that most of candidate sequences have duplications. So I filtered them out based on 'Self' mapping information from MUSCLE output (if a sequence is mapped itself longer than 50 bp). Finally 10 fragments are selected.
- hoxa3 - XENTR_CHORI-219_ID.24.muscle, XENTR_CHORI-219_ID.25.muscle, XENTR_CHORI-219_ID.26.muscle
- pitx-1 - XENTR_CHORI-219_ID.27.muscle, XENTR_CHORI-219_ID.28.muscle, XENTR_CHORI-219_ID.29.muscle
- hoxa5 - XENTR_CHORI-219_ID.2.muscle, XENTR_CHORI-219_ID.3.muscle
$ mus4 -i XENTR_CHORI.fasta -o XENTR_CHORI.muscle
Here's the example of Self duplication.
>XENTR_NM_001113032_18 gi|163915026|ref|NM_001113032| 3 + ccaggggtacccagggcacaaataagcactcaccccaaatccccccctaactggccttcaggct 66 ||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||| 138 + ccaggggtacccagggcacaaataagcactcaccccaaatctccccctaactggccttcaggct 201 67 + gggcccccttagcccataacaaggttacagatagttagaaacattggg 114 ||||||||||||||||||||||||||||||||| ||||||||||||| 202 + gggcccccttagcccataacaaggttacagatatatagaaacattggg 249