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## |5| Dotplots and Sequence Alignments
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## Dot Plots

# Nucleic Acid Dot Plots using the Molecular Toolkit (http://arbl.cvmbs.colostate.edu/molkit/)

a1) Generate a random DNA sequence with GC content of 0.5 (http://www.faculty.ucr.edu/~mmaduro/random.htm)
a2) Copy paste the sequence into both windows, choose windows size of 9, and click [Make Plot]
a3) Generate a new random DNA sequence with GC content of 0.1 and repeat step a1-a3
a4) Change windows size to 5 and mismatch to 1 
a5) Change windows size to 11 and mismatch to 2 and compare the plot with a5

# Nucleic Acid Dot Plots using Dotlet (http://myhits.isb-sib.ch/cgi-bin/dotlet)

(b1) [Input]: Upload the following four sequences 

>S1 P05049 SNAK_DROME Serine protease snake OS=Drosophila melanogaster GN=snk PE=1 SV=2
MIILWSLIVHLQLTCLHLILQTPNLEALDALEIINYQTTKYTIPEVWKEQPVATIGEDVD
DQDTEDEESYLKFGDDAEVRTSVSEGLHEGAFCRRSFDGRSGYCILAYQCLHVIREYRVH
GTRIDICTHRNNVPVICCPLADKHVLAQRISATKCQEYNAAARRLHLTDTGRTFSGKQCV
PSVPLIVGGTPTRHGLFPHMAALGWTQGSGSKDQDIKWGCGGALVSELYVLTAAHCATSG
SKPPDMVRLGARQLNETSATQQDIKILIIVLHPKYRSSAYYHDIALLKLTRRVKFSEQVR
PACLWQLPELQIPTVVAAGWGRTEFLGAKSNALRQVDLDVVPQMTCKQIYRKERRLPRGI
IEGQFCAGYLPGGRDTCQGDSGGPIHALLPEYNCVAFVVGITSFGKFCAAPNAPGVYTRL
YSYLDWIEKIAFKQH

>S2 P08246 ELNE_HUMAN Neutrophil elastase OS=Homo sapiens GN=ELANE PE=1 SV=1
MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHAWPFMVSLQLRGGHFCGATLI
APNFVMSAAHCVANVNVRAVRVVLGAHNLSRREPTRQVFAVQRIFENGYDPVNLLNDIVI
LQLNGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRNRGIASVLQELNVTVVTSL
CRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVN
WIDSIIQRSEDNPCPHPRDPDPASRTH

>S3 Q9P255 ZN492_HUMAN Zinc finger protein 492 OS=Homo sapiens GN=ZNF492 PE=2 SV=2
MLENYRNLVFVGIAASKPDLITCLEQGKEPWNVKRHEMVAEPPVVCSYFARDLWPKQGKK
NYFQKVILRRYKKCGCENLQLRKYCKSMDECKVHKECYNGLNQCLTTTQNKIFQCDKYVK
VFHKFSNSNRHTIRHTGKKSFKCKECEKSFCMLSHLAQHKRIHSGEKPYKCKECGKAYNE
TSNLSTHKRIHTGKKPYKCEECGKAFNRLSHLTTHKIIHTGKKPYKCEECGKAFNQSANL
TTHKRIHTGEKPYKCEECGRAFSQSSTLTAHKIIHAGEKPYKCEECGKAFSQSSTLTTHK
IIHTGEKFYKCEECGKAFSQLSHLTTHKRIHSGEKPYKCEECGKAFKQSSTLTTHKRIHA
GEKFYKCEVCSKAFSRFSHLTTHKRIHTGEKPYKCEECGKAFNLSSQLTTHKIIHTGEKP
YKCEECGKAFNQSSTLSKHKVIHTGEKPYKYEECGKAFNQSSHLTTHKMIHTGEKPYKCE
ECGKAFNNSSILNRHKMIHTGEKLYKPESCNNACDNIAKISKYKRNCAGEK

>S4 P21997 SSGP_VOLCA Sulfated surface glycoprotein 185 OS=Volvox carteri PE=1 SV=1
MSKLLLVALFGAIAVVATSAEVLNLNGRSLLNNDDPNAFPYCKCTYRQRRSPYRLKYVGA
ENNYKGNDWLCYSIVLDTTGTVCQTVPLTEPCCSADLYKIEFDVKPSCKGTVTRAMVFKG
IDRTVGGVRVLESISTVGIDDVTGVPGAAILRIVKDLALPYSVVASFLPNGLPVCINRVP
GSCTFPELFMDVNGTASYSVFNSDKDCCPTGLSGPNVNPIGPAPNNSPLPPSPQPTASSR
PPSPPPSPRPPSPPPPSPSPPPPPPPPPPPPPPPPPSPPPPPPPPPPPPPPPPPPSPSPP
RKPPSPSPPVPPPPSPPSVLPAATGFPFCECVSRSPSSYPWRVTVANVSAVTISGGAGER
VCLKISVDNAAAATCNNGLGGCCSDGLEKVELFANGKCKGSILPFTLSNTAEIRSSFSWD
STRPVLKFTRLGLTYAQGVAGGSLCFNIKGAGCTKFADLCPGRGCTVAVFNNPDNTCCPR
VGTIA

(b2) Compare sequence #1 with itself
(b3) Compare sequence #3 with itself (windows size 21, zoom 1:2)
(b4) Compare sequence #4 with itself (windows size 7, zoom 1:2)
(b5) Compare sequence #1 with #2 (windows size 51, zoom 1:2)

# Sequence comparison:

(c1) Design a sequence with an inverted region in the middle and compare it with it self using dot plot approach.
     You might use the Molecular Toolkit to create the sequence (http://arbl.cvmbs.colostate.edu/molkit/).


## Alignments


(a) Calculate "identity" and "similarity" for the two sequences below. Use a binary (0/1) matrix and
	 apply a -1 penalty for gaps. (Hit: you could use a spreadsheet application like Excel)

	>SeqWordA
	THECATISINTHEHOUSE
	>SeqWordB
	THETWOCATSAREONTHEHOUSE


# For the next exercises you might use one of the following web based alignment applications:

	Pairwise: 	LALIGN (http://www.ch.embnet.org/software/LALIGN_form.html)
	MSA: 		ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/)
				MAFFT (http://www.ebi.ac.uk/Tools/msa/mafft/)

(b2) Align the two DNA sequences - How many of the 111 sites are identical?

	>Seq_2b1
	TGTAGTAGAGGCGACGCGGGTGCGGTCATCACTAATAAGGATATTAATCAACATAGTAGAAAAACTCACAGGCCTCCGCCTTTAGGCGGTGCTTACTCTTAC
	>Seq_2b2
	TGTAGTAGAAGGCGACGCGGGTGCGGTCATCACTAATAAGGATATTAATAAACAGCACAGCAAGTTGTCAAAAACTCACAGGCCTCCGCCTTTAGGCGGTGCTTACTCTTAC

(b3) Translate the DNA into a protein sequence and re-align the sequences. Is the alignment better? What happen?
(b4) Try to find a way to get a better protein alinment.

(c) Global vs. local alignment

(c1) What is better, global or local alignments?

(c2) Use the following two sequences and perfom a local and a global alignment. You can use LALIGN for the alignments.

	http://www.ch.embnet.org/software/LALIGN_form.html

	>Seq_2c1
	GCGATTTAGCGTGACAGCCCCAGGGAACCCACAAAATGTGATCGCAGTCCATCCGATCGTACACAGAAAGGAAGGTCCCCATACACCGACGCACCTGTTT
	>Seq_2c2
	ACACGTCGTATGCATAAACGAGCCGCACGAACCAGAGAGCATAAAGAGGACCTCTAGTTCTGACAGCCCCAGGGAACCCACAAAATGCCAAGATGCCTTA

(d1) Align the following four protein sequences. Do these sequences have something in common?

	>PS_2d1
	DGVPARQVILDTCGARRDDMSAALQCSNSMQRQINLKGRVIAVGKSRGLPKRKYTNPAINYRKSFKGWAFQVIYFGFSAIGELELSLLLIIGPRDEA
	>PS_2d2
	YLSDSPQEFAMQGYVLLFGQGISYPAKELKADVEKPARQVILDTCGARRDDMSAAFIHARTTSSAGCKKVERWNSPARLSRFIVKPNIPR
	>PS_2d3
	AAFAIKESEELEGSKIVCDPVGGHGPVFRDSSELFRAKNVPELALVYKTQAGQYLLLEMNIPARQVILDTCGARRDDMSAARADKFDGTRSLTNGIVRLP
	>PS_2d4
	EDKQQEPARQVILDTCGARRDDMSAARPHENVPVETAGFVKSGTWSLSFLKFSRGITLSLVEDLLEPAISQNAFSGLQANKVMVDHLDELTYDY

(d2) Generate a sequence logo with the aligned protein sequences using Weblogo (http://weblogo.berkeley.edu/logo.cgi)