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EMBOSS utilities

1. commandline
   1. on perun
      1. conda environment
         1. source activate emboss
2. online graphical interface

https://emboss.bioinformatics.nl/cgi-bin/emboss/

1. also includes documentation

https://www.ebi.ac.uk/services/all

1. doesn’t seem to have all of them
2. local install (good luck with that)

1. common syntax
   1. help
   2. sequence
   3. outfile
   4. outseq

Name_of_multifasta_file:name_of_individual_fasta

1. auto

1. lots of output file formats

embl, genbank, gff, pir, swiss

How to change lowercase to upper case

more p12

>seqW
AGGGTGTGGTGCCCGGccccttt
>tok3
Ccgtgaccgggatact

maskfeat -sequence p12 -supper1 -outseq p12upper

>seqW
AGGGTGTGGTGCCCGGCCCCTTT
>tok3
CCGTGACCGGGATACT

How to sort a multifasta file based on size of individual sequences

1. can be largest or smallest first

more p15

>M86863.1
GTAACATGACGTTGACC
>seq15
ACTCGGGGGCGGAGTGGGTCACACTTTCCTTTACTCGGGGGCGGAGTGGGTCACGTGACTTTCCTTT
>seq2
ACTCGGGGGCGGAGTGGGTCACGTGACTTTCCTTT
>NINA
TTTA

sizeseq -sequence p15 -outseq p15sized -descending N

more p15sized

>NINA
TTTA
>M86863.1
GTAACATGACGTTGACC
>seq2
ACTCGGGGGCGGAGTGGGTCACGTGACTTTCCTTT
>seq15
ACTCGGGGGCGGAGTGGGTCACACTTTCCTTTACTCGGGGGCGGAGTGGGTCACGTGACT
TTCCTTT
	-note that the output is interleaved
	-to reverse size order -descending Y

How to get basic info about the individual fastas

1. length
2. GC%

infoseq p2.fasta -length -only Display basic information about sequences Length 25 54 32 32 25

1. if you want to save the information -outfile name_of_file
2. can just do infoseq p2.fasta

How to reverse and/or complement fastas

revseq

1. can do just reverse, just complement or both

More p7.fasta

>seq1
AAAAAAAAAAGGGGGGGGGG
>seq2
AGAGAGTT

revseq -sequence p7.fasta -outseq p7justcomplemented -reverse N Or revseq -sequence p7.fasta -outseq p7justcomplemented -noreverse

>seq1
TTTTTTTTTTCCCCCCCCCC
>seq2
TCTCTCAA

revseq -sequence p7.fasta -outseq p7justrev -nocomplement or revseq -sequence p7.fasta -outseq p7justreve -complement N

>seq1
GGGGGGGGGGAAAAAAAAAA
>seq2
TTGAGAGA

To both reverse and complement revseq -sequence p7.fasta -outseq p7rcemboss

>seq1 Reversed:
CCCCCCCCCCTTTTTTTTTT
>seq2 Reversed:
AACTCTCT

If you don’t want the string Reversed: in the header put -notag or -tag N

How to split a multifasta into individual fastas I like to make a separate directory to hold the individual fastas but it is not necessary. mkdir -m 777 SPLIT_GENOME mv genome SPLIT_GENOME cd SPLIT_GENOME/ grep “>” genome -c 68

Fastest way is seqretsplit genome -auto

1. auto will turn off prompting for file names
2. .fasta will be added to the name of each individual fasta file

which is derived from the header

1. will make the name of the file lower case
   1. the header remains the same
2. will make the lines 60 characters wide
3. little control over names of individual files
4. osextension2 string

How to do an amino acid translation of a multifasta

transeq
	-will simply translate the sequences and insert * for stops
	-can change *s to Xs
		-clean
	-can translate different frames
		-frame 1 is default
			-frame 1
			-frame 2
			-frame 3
			-frame F
			-frame -1
			-frame -2
			-frame -3
			-frame R
			-frame 6 
	-can use different genetic codes
		-table 0 (standard and default)
		-table  2 (Vertebrate mitochondrial)
		-table 11 (Bacterial)
	-can designate a range or region otherwise entire thing
		-regions 24-100

1. output is sequence-name_frame-number

Eg >seqA_4 more p11

>seqA
CCCTGGCGATTAAGCCAGACATTAGTCCGGGGCATCCCAGCTCGTGATTTCAGGGGCGAC
CACTTTCGAAAATGGGCACGACCCAGCGCAACGAGTTGGAACGCTATCGCCGGGAGATTG
GCTTAATCGCGAGCGCTAGCTAGGCACTAGCGATCTAGGGGATGTGCCGACTGTAGCTCC
>seqB
TCCAACATGCCGACCCGTTCCATCCACCACAAGACCGAGACCACTGTCGTCTTCGCGGAAAGCCCGCCCATGCGCCCCGGCTCGCTCGCGCTCGTCGTAGTCAACAGAGT
GCATACACGGTCCTTGCGGCAGACGAAGGTCCCAGTGGCCGTGATGTCGCCCCGTGCTTCCCAGACGACCGACCTGCTTGCTGACTGGGCGATTGTTGTCATCCAGTACC
TCGAGGATCTTTTCGGGGTCGAGTTCCCCATCCCAAAGCTCGACATCGTCTTCCTCCCCGAGTTCCCCGTGTCCGCCTTTGAGAACTTCGGCTGCATCCTGTTCAGAAAG
ACAACCGCCCGACTCCCGACTCTTTTTTCGACTGTGGCGCACGAGATCATCCACCAGTACTTTGGGCAGGGTGTAGGCCACGCCAGGGCCCATGAGACGTTCCTTGCTGA
GGGCCCCGCCCGTTTCTTCCAGTACAAGGTCATGGCCGACTGCTTCGAGGAGTTCAACAGAGCCCATAGCCTCGAAAGACAGCTGCCGGACTTGGCAGCGAAGTTCTACA
TTGATGTCATCGATGCAGGCCTGGCTAAGGAGACTGCGTGTCCGCCCTTTACACTGGGGCTCACTCCAGACGTCAGGGTCGAGCGGCGCGAGGGGGCATATGTCACTGTA
GTCGGGGCAGAGATTTGTCAGAATGACGCATTCTATGACGATTTGGTCTATACAAAGGGCGCGGCGCTGTTTCATATGATCGCAGGTCTGTTTCCCGCGACAGCTGGGCA
CAATCCGTTCATTCGTGCGCTGTCAGCATACTTATCTGACAACATGTTCTCCGATGTCACTAGCGAGACGTTAATTTCGTATTTAACGAAGCTGAGACACCCAGAGATTC
CCGCTCAGATCATTACGAAGCTGATCCACGACCACATCAACCTACCCCTGTTCCCGACTGTAGCAGCGTCGGTTGTACCCATCGCAGAC

	transeq -sequence p11 -outseq wha -frame 1,2
>seqA_1
PWRLSQTLVRGIPARDFRGDHFRKWARPSATSWNAIAGRLA*SRALARH*RSRGCADCSS
>seqA_2
PGD*ARH*SGASQLVISGATTFENGHDPAQRVGTLSPGDWLNRER*LGTSDLGDVPTVAP
>seqB_1
SNMPTRSIHHKTETTVVFAESPPMRPGSLALVVVNRVHTRSLRQTKVPVAVMSPRASQTT
DLLADWAIVVIQYLEDLFGVEFPIPKLDIVFLPEFPVSAFENFGCILFRKTTARLPTLFS
TVAHEIIHQYFGQGVGHARAHETFLAEGPARFFQYKVMADCFEEFNRAHSLERQLPDLAA
KFYIDVIDAGLAKETACPPFTLGLTPDVRVERREGAYVTVVGAEICQNDAFYDDLVYTKG
AALFHMIAGLFPATAGHNPFIRALSAYLSDNMFSDVTSETLISYLTKLRHPEIPAQIITK
LIHDHINLPLFPTVAASVVPIAD
>seqB_2
PTCRPVPSTTRPRPLSSSRKARPCAPARSRSS*STECIHGPCGRRRSQWP*CRPVLPRRP
TCLLTGRLLSSSTSRIFSGSSSPSQSSTSSSSPSSPCPPLRTSAASCSERQPPDSRLFFR
LWRTRSSTSTLGRV*ATPGPMRRSLLRAPPVSSSTRSWPTASRSSTEPIASKDSCRTWQR
SSTLMSSMQAWLRRLRVRPLHWGSLQTSGSSGARGHMSL*SGQRFVRMTHSMTIWSIQRA
RRCFI*SQVCFPRQLGTIRSFVRCQHTYLTTCSPMSLARR*FRI*RS*DTQRFPLRSLRS
*STTTSTYPCSRL*QRRLYPSQT

transeq -sequence p11 -outseq wha -frame 1,2 -clean

>seqA_1
PWRLSQTLVRGIPARDFRGDHFRKWARPSATSWNAIAGRLAXSRALARHXRSRGCADCSS
>seqA_2
PGDXARHXSGASQLVISGATTFENGHDPAQRVGTLSPGDWLNRERXLGTSDLGDVPTVAP
>seqB_1
SNMPTRSIHHKTETTVVFAESPPMRPGSLALVVVNRVHTRSLRQTKVPVAVMSPRASQTT
DLLADWAIVVIQYLEDLFGVEFPIPKLDIVFLPEFPVSAFENFGCILFRKTTARLPTLFS
TVAHEIIHQYFGQGVGHARAHETFLAEGPARFFQYKVMADCFEEFNRAHSLERQLPDLAA
KFYIDVIDAGLAKETACPPFTLGLTPDVRVERREGAYVTVVGAEICQNDAFYDDLVYTKG
AALFHMIAGLFPATAGHNPFIRALSAYLSDNMFSDVTSETLISYLTKLRHPEIPAQIITK
LIHDHINLPLFPTVAASVVPIAD
>seqB_2
PTCRPVPSTTRPRPLSSSRKARPCAPARSRSSXSTECIHGPCGRRRSQWPXCRPVLPRRP
TCLLTGRLLSSSTSRIFSGSSSPSQSSTSSSSPSSPCPPLRTSAASCSERQPPDSRLFFR
LWRTRSSTSTLGRVXATPGPMRRSLLRAPPVSSSTRSWPTASRSSTEPIASKDSCRTWQR
SSTLMSSMQAWLRRLRVRPLHWGSLQTSGSSGARGHMSLXSGQRFVRMTHSMTIWSIQRA
RRCFIXSQVCFPRQLGTIRSFVRCQHTYLTTCSPMSLARRXFRIXRSXDTQRFPLRSLRS
XSTTTSTYPCSRLXQRRLYPSQT

sixpack
	-more output option than transeq
	-tries to find orfs in a sequence
	-ONLY TAKES ONE SEQUENCE
	-generates two files
		-outseq=protein sequences
		-outfile=graphical six frame translation and numbers of 
		ORFs per frame

First example with just one sequence

more p10

>seqA
CCCTGGCGATTAAGCCAGACATTAGTCCGGGGCATCCCAGCTCGTGATTTCAGGGGCGAC
CACTTTCGAAAATGGGCACGACCCAGCGCAACGAGTTGGAACGCTATCGCCGGGAGATTG
GCTTAATCGCGAGCGCTAGCTAGGCACTAGCGATCTAGGGGATGTGCCGACTGTAGCTCC

sixpack -sequence p10 -outseq p10.prot.fa -outfile p10.stuff

>seqA_1_ORF1  Translation of seqA in frame 1, ORF 1, threshold 1, 41aa
PMRLSQTLVRGIPARDFRGDHFRKWARPSATSWNAIAGRLA
>seqA_1_ORF2  Translation of seqA in frame 1, ORF 2, threshold 1, 7aa
SRALARH
>seqA_1_ORF3  Translation of seqA in frame 1, ORF 3, threshold 1, 10aa
RSRGCADCSS
>seqA_2_ORF1  Translation of seqA in frame 2, ORF 1, threshold 1, 3aa
PCD
>seqA_2_ORF2  Translation of seqA in frame 2, ORF 2, threshold 1, 3aa
ARH
>seqA_2_ORF3  Translation of seqA in frame 2, ORF 3, threshold 1, 37aa
SGASQLVISGATTFENGHDPAQRVGTLSPGDWLNRER
>seqA_2_ORF4  Translation of seqA in frame 2, ORF 4, threshold 1, 14aa
LGTSDLGDVPTVAP
>seqA_3_ORF1  Translation of seqA in frame 3, ORF 1, threshold 1, 14aa
HAIKPDISPGHPSS
>seqA_3_ORF2  Translation of seqA in frame 3, ORF 2, threshold 1, 31aa
FQGRPLSKMGTTQRNELERYRREIGLIASAS
>seqA_3_ORF3  Translation of seqA in frame 3, ORF 3, threshold 1, 4aa
ALAI
>seqA_3_ORF4  Translation of seqA in frame 3, ORF 4, threshold 1, 5aa
GMCRL
>seqA_3_ORF5  Translation of seqA in frame 3, ORF 5, threshold 1, 2aa
LX
>seqA_4_ORF1  Translation of seqA in frame 4, ORF 1, threshold 1, 14aa
GATVGTSPRSLVPS
>seqA_4_ORF2  Translation of seqA in frame 4, ORF 2, threshold 1, 37aa
RSRLSQSPGDSVPTRCAGSCPFSKVVAPEITSWDAPD
>seqA_4_ORF3  Translation of seqA in frame 4, ORF 3, threshold 1, 3aa
CLA
>seqA_4_ORF4  Translation of seqA in frame 4, ORF 4, threshold 1, 3aa
SHG
>seqA_5_ORF1  Translation of seqA in frame 5, ORF 1, threshold 1, 7aa
SYSRHIP
>seqA_5_ORF2  Translation of seqA in frame 5, ORF 2, threshold 1, 4aa
IASA
>seqA_5_ORF3  Translation of seqA in frame 5, ORF 3, threshold 1, 11aa
LALAIKPISRR
>seqA_5_ORF4  Translation of seqA in frame 5, ORF 4, threshold 1, 17aa
RSNSLRWVVPIFESGRP
>seqA_5_ORF5  Translation of seqA in frame 5, ORF 5, threshold 1, 17aa
NHELGCPGLMSGLIAWX
>seqA_6_ORF1  Translation of seqA in frame 6, ORF 1, threshold 1, 10aa
ELQSAHPLDR
>seqA_6_ORF2  Translation of seqA in frame 6, ORF 2, threshold 1, 7aa
CLASARD
>seqA_6_ORF3  Translation of seqA in frame 6, ORF 3, threshold 1, 41aa
ANLPAIAFQLVALGRAHFRKWSPLKSRAGMPRTNVWLNRMG

Refine the output

sixpack -sequence p10 -outseq p10.prot.fa -outfile p10.stuff -nofirstorf -nolastorf -orfminsize 30

>seqA_1_ORF1  Translation of seqA in frame 1, ORF 1, threshold 30, 41aa
PMRLSQTLVRGIPARDFRGDHFRKWARPSATSWNAIAGRLA
>seqA_2_ORF1  Translation of seqA in frame 2, ORF 1, threshold 30, 37aa
SGASQLVISGATTFENGHDPAQRVGTLSPGDWLNRER
>seqA_3_ORF1  Translation of seqA in frame 3, ORF 1, threshold 30, 31aa
FQGRPLSKMGTTQRNELERYRREIGLIASAS
>seqA_4_ORF1  Translation of seqA in frame 4, ORF 1, threshold 30, 37aa
RSRLSQSPGDSVPTRCAGSCPFSKVVAPEITSWDAPD

sixpack -sequence p10 -outseq p10.prot.fa -outfile p10.stuff -nofirstorf -nolastorf -orfminsize 30 -mstart Y

>seqA_1_ORF1  Translation of seqA in frame 1, ORF 1, threshold 30, 40aa
MRLSQTLVRGIPARDFRGDHFRKWARPSATSWNAIAGRLA

How to do multiple fastas? mkdir -m 777 SPLIT cp p11 SPLIT cd SPLIT seqretsplit p11 -auto

1. generates individual fastas

ls *fasta > list

1. if necessary remove the multifasta file name from list

for i in `cat list`; do sixpack -sequence $i -outfile $i.stuff -outseq $i.prot; done

can collect all the outputs together if you want cat *prot >alloutputs

How to extract regions

more p2.fasta

>sequence1 blahblah
ACGTACGTACGTACGTACGTACGTT
>sequence47 bluhbluhbluh
TTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCC
>myfavourite
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>myfav2
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>contig23 acanth
AGTAGTGACTGAGTAATAGACGTAG

extractseq p2.fasta:myfav2 -reg “1-5” -outseq blah2

>myfav2
AGAGA

extractseq p2.fasta:myfav2 -reg “1-5 7-9” -outseq blah2 -separate

>myfav2_1_5
AGAGA
>myfav2_7_9
AGA

How to extract a subset of fastas from a multifasta See http://129.173.88.134:81/dokuwiki/doku.php?id=extracting_a_single_fasta_entry_or_multiple_from_a_multifasta_file

seqret

more p2.fasta

>sequence1 blahblah
ACGTACGTACGTACGTACGTACGTT
>sequence47 bluhbluhbluh
TTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCC
>myfavourite
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>myfav2
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>contig23 acanth
AGTAGTGACTGAGTAATAGACGTAG

Want to extract just myfav2 and sequence47

Make a list with the sequences you want like this Name_of_multifastafile:name_of_individual_file1 Name_of_multifastafile:name_of_individual_file1

SO, more filestoget p2.fasta:myfav2 p2.fasta:sequence47

Then seqret @filestoget -outseq myfavouritefiles -auto

more myfavouritefiles

>myfav2
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>sequence47 bluhbluhbluh
TTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCC

If there is a common and unique string to the header ids you can use that

seqret p2.fasta:my* -outseq yup -auto

more yup

>myfavourite
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
>myfav2
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG