The document describes analyzing nucleotide sequences of the rhodopsin gene from human, chimpanzee and macaque. Key steps include: 1) Obtaining rhodopsin coding sequences from NCBI and writing them to a FASTA file 2) Performing a multiple sequence alignment using ClustalW 3) Calculating the transition/transversion ratio and genetic distance between species based on the alignment

1. 2010/06/17
kaneko.satoko(at)ocha.ac.jp

2.
R /R
R Bioconductor(GeneR)

3. ( )

4. – ‐
( )
( )
( )

5. – ‐
(A:61%, G:32%, C: 7%)
5 (A:80%, G:20%) 10 (A:60%, G:40%)
5
01 A 10
( )
02 A
03 A 01 A
A G A G G 02 A
G A A 04 G
A A A G 05 A 03 A
G A A C G 04 G
C A A A: 80%, G: 20%
A A A A 05 A
A G A G
06 G
07 A
08 G
09 A
10 G
A: 60%, G: 40%

6. – ‐
1 n
x = ∑ xi
n i=1
( )
€
( ‐1) ( )

7. – ‐
5
5, 0.5
5, 1.0
5, 2.0

8. R
R
R ( )
Bioconductor(GeneR)

9. R
R
(2010 6 14 R 2 11.1)
R‐2.11.1.dmg

10. R – ‐
R
q()
Save workspace image? [y/n/c]:
y
n

11. R –R ‐
R dock

12. R – ‐

13. R – ‐
> 1+2 #
[1] 3
+
>3‐1 # ‐
[1] 2
*
>10*2 # /
[1] 20 ^
>7/2 # sqrt()
[1] 3.5
> 2^2 #
[1] 4
> sqrt(4) #
[1] 2

14. R – ‐
R
c
> x <‐ c(1,2,5,6) # c() () x
> x
[1] 1 2 5 6
> x + 10 #x (1,2,5,6) 10
[1] 11 12 15 16
> x * 10 #x 10
[1] 10 20 50 60
> x <‐ c(“France”, “123”, “abc”) # “”
> x
[1] "France" "123" "abc"

15. R – ‐
> number<‐ c(1,2,3,4,5,6,7,8,9,10)
> number
[1] 1 2 3 4 5 6 7 8 9 10
> length (number) #length
[1] 10
> sum (number) #
[1] 55
> max (number) #
[1] 10
>min (number) #
[1] 1
> mean(number) #
[1] 5.5

16. R – 1‐
[41,55,51,61,45,38,60,48,43,46,63,51,55,55,53]
> x<‐ c(41,55,51,61,45,38,60,48,43,46,63,51,55,55,53)
> length(x)
[1] 15
> sum(x) #
[1] 765
#
> 765/15
> mean(x)
[1] 51

17. R – 2‐
[41,55,51,61,45,38,60,48,43,46,63,51,55,55,53]
> sub <‐ x‐51
> sub
[1] ‐10 4 0 10 ‐6 ‐13 9 ‐3 ‐8 ‐5 12 0 4 4 2
> sub^2
[1] 100 16 0 100 36 169 81 9 64 25 144 0 16 16 4
> sum(sub^2)
[1] 780
> 780/15
[1] 52
>sqrt(52) #
[1] 7.211103

18. Bioconductor
h`p://www.bioconductor.org/packages/release/Soaware.html

19. Bioconductor/GeneR
GeneR
> source("h`p://www.bioconductor.org/biocLite.R")
> biocLite("GeneR")
> library(GeneR)
> ls(“package:GeneR”) #GeneR

20. Bioconductor/GeneR 1
> s<‐"gtcatgcatgctaggtgacag`aaaatgcgtctaggtgacagtctaacaa"
> placeString(s) #GeneR placeString()
[1] 0
> translate() # 1
[1] "VMHAR*QLKCV*VTV*Q"
> translate (from=c(1,2,3),to=c(0,0,0)) #3
[1] "VMHAR*QLKCV*VTV*Q" "SCMLGDS*NASR*QSN" "HAC*VTVKMRLGDSLT"
> strCompoSeq(s, wsize=1) # (% )
T C A G X
[1,] 0.2549020 0.1764706 0.3137255 0.2549020 0
> strCompoSeq(s, wsize=2) # 2
TT TC TA TG TX CT CC CA CG CX AT AC AA AG AX GT GC
[1,] 0 0 0.04 0.08 0 0.12 0 0.2 0 0 0.04 0 0.08 0.08 0 0.24 0.04
GA GG GX XT XC XA XG XX
[1,] 0.08 0 0 0 0 0 0 0

21. ‐Rhodopsin ‐
Rhodopsin (RHO)
5 exons, CDS 1047bp (348a.a.)
human, chimpanzee, macaque Rhodopsin
transioon, transversion
1) REST
2) CDS R CDS
3) ClustalW mulople alignment
4) transioon transversion

22. Clustalw
Clustalw mulople alignment
alignment output format
Clustalw Clustalx GUI
available for download here

23. Clustalw
Clustalw‐2.0.12 (2010 6 )
‐2.0.12‐
‐2.0.12‐
Users/tg03/bin
ap.ebi.ac.uk
clustalw‐2.0.12macosx

24. (nucleoode) A,T,G,C 4
A,G purine T,C pyrimidine
A T G C
purine pyrimidine transioon
purine pyrimidine transversion
C
α
T
β
β
β
β
A
T
α
A
G
:transioon
α
:transversion
β
G
C
purine
pyrimidine
Figure 4‐4 Molecular Biology of the Cell (© Garland Science 2008)

25. transioon/transversion
Rhodopsin CDS(coding sequence) 1047bp
transioon: A G, C T
transversion: A,G C,T
NCBI ID
human(NM_000539) chimpanzee(XM_516740) macaque(XM_001094250)
human – chimpanzee, human – macaque
transioon transversion
p distace
p distance = /
transioon
transversion
p distance
human‐
chimpanzee
human‐
macaque

26. Bioconductor/GeneR 1
R Users/tg03/bin
> library(GeneR) # GeneR R
1) REST NCBI Rhodopsin
x R
> x <‐ " "
> s <‐placeString(x)
2) CDS R CDS
# rhodopsin.txt fasta
> writeFasta(file="rhodopsin.txt",from =96, to=1142, comment="human")
[1] 1 # 1: , ‐1:

27. Bioconductor/GeneR 2
1’) REST NCBI chimpanzee Rhodopsin
> x <‐ " "
> s <‐placeString(x)
2’) CDS R CDS
> writeFasta(file=“rhodopsin.txt”, from = , to= , comment= "chimpanzee", append=T)
#append=T
1’’) REST NCBI macaque Rhodopsion
> x <‐ " "
> s <‐placeString(x)
2’’) CDS R CDS
> writeFasta(file=“rhodopsin.txt”, from = , to= , comment= "macaque", append=T)
Users/tg03/bin rhodopsin.txt
Seq_R_96_1142

28. Clustalw
3) ClustalW mulople alignment
$ export PATH=/Users/tg03/bin/clustalw‐2.0.12‐macosx
$ cd /Users/tg03/bin/clustalw‐2.0.12‐macosx
$mv ~/bin/rhodopsin.txt ~/bin/clustalw‐2.0.12‐macosx
$ clustalw2 ‐INFILE=rhodopsin.txt ‐OUTFILE=rho.aln
rho.aln
CotEditor / Courier Courier New
4) transioon transversion

29.
Rhodopsin CDS(coding sequence) 1047bp
transioon: A G, C T
transversion: A,G C,T
human – chimpanzee, human – macaque
(NM_000539)‐(XM_516740) (NM_000539)‐(XM_001094250)
transioon transversion
transioon
transversion
p distance
human‐
5
1
6/1047 = 0.006
chimpanzee
human‐
32
8
40/1047 = 0.038
macaque
600
3000

30.
2
2
1
2
TCTGAGACCT
TCTGTGACCT
5th A T 6th G C
3th T A 6th G C
6th G C
3th T A
9th C G 9th C G
5th A T 6th C G
TCAGTGACCT TCTGTCACGT TCAGTGACCT TCTGTCACGT

31.
1) p distance:
: ,
:
2) Jukes and Cantor model (1969): A
T
C
G
A
‐
α
α
α
( ) [ ( )]
d = − 3 4 ln 1− 4 3 p T
α
‐
α
α
G
α
α
‐
α
3) Kimura’s two parameter model (1980):
C
α
α
α
‐
transioon
€ transvesion
A
T
C
G
( )(
P = 1 4 1− 2e−4 (α + β ) t + e−8 βt ) A
‐
β
β
α
T
β
‐
α
Q= 1( 2)(1− e )−8 βt
G
β
α
‐
β
d ≡ 2rt = 2αt + 4 βt
€ C
α
β
β
‐
€
( ) ( )
= − 1 2 ln(1− 2P − Q) − 1 4 ln(1− 2Q)

32. 1
( a) a’,a”
t t+1
a’
t
t+1
T a’
T a’
T a’
(1‐r)
(1‐r)2
a
A
G
C
T a
a’’
1‐2r
(1‐r)
T a”
T a”
T a”
r 10‐8,10‐9
A
G
C
r =3α
r2
(
t T a’
T a’
(1‐r)
)
a”
:r
C a”
T a”
r/3
2r(1‐r)/3
1‐r
t A a’
T a’
r/3
2r/3
a’
:1/3
T a”
T a”
(1‐r)

33. 2
Jukes and Cantor model (1969):
t t+1
qt qt+1
qt t qt+1‐qt dq/dt
(d)
2rt d
qt
( ) [ ( )]
d = − 3 4 ln 1− 4 3 p pt=(1‐qt):
p:
(p distance)
€

34. human, chimanzee, macaque Rhodopsin
Jukes and Cantor model
transioon
transversion
p distance
JC distance
human‐
5
1
6/1047 = 0.006
chimpanzee
human‐
32
8
40/1047 = 0.038
macaque
( ) [ ( )]
d = − 3 4 ln 1− 4 3 p
4
€ > ‐(3/4)*log(1‐(4/3)*0.006)

35. R
(transioon/transversion )