Post on 17-Jun-2020
PCR of rbcL in Scutellaria sp.
S. st
rigill
osa
1
S. st
rigill
osa
2
S. peki
nensi
s va
r. a
lpin
a
S. in
dic
a
Isodon inflexs
us
500bp
1,000bp
1,500bp
DNA sequencing
Sanger methodSanger and Gilbert shared a Nobel prize in 1980
Classical one-dye (or isotope) four-lane systemhttp://www.youtube.com/watch?v=6ldtdWjDwes
Acrylamide Gel Electrophoresis with S35 for Sequencing
Gel dryer
IntensifyingCacette
Four-dye one-lane system using four different florescence dyes
1990’~2000’ABI 377: Gel-type Automatic sequencer
Current sequencing service machine ABI 3730: Capillary-type Automatic sequencer
ABI 3100
http://www.youtube.com/watch?v=SRWvn1mUNMA&feature=related
Next generation sequencing (NGS)Pyro-Sequencing
1) Emulsion PCR2) Sequencing by synthesis (SBS)3) Microtiter plate
Three Big Companies
Roche GS-FLX 100M bp / runIllumina Solexa 80G bp X 4 /runABI SOLid 200G bp /run
50 Million Clusters Per Flow Cell
20 Microns
100 Microns
Solexa Sequencing
Sequencing request to the service company:e.g., MACROGEN
Input order information
Input sample and primerinformation
Download sequencing result
RESULT
Sequence editing using SEQUENCHER
Open .ab1 files
Sequence of one “.ab1” files
Chromatogram of one “.ab1” files
rbcLAssemble four sequences from four reactions
A “Contig” is generated rbcL
Heterogeneous parts should be “edited”
Now heterogeneous parts are “edited”
“Primer” sequences should be deleted.
Save edited sequencesas text file with FASTA format
FASTA format:
>taxa name1sequence…
>taxa name2sequence…
>taxa name3sequence…
…
Sequence comparison and phylogenetic analysis using MEGAOpen FASTA file in the MEGA
Translate DNA sequences into amino-acid sequences.
Adjust “frame”!
Do sequence “alignment”
Now sequences are well aligned
Change aligned AA sequences into DNA sequences
Now it is ready to do analyses Change the matrix into MEGA format
Result of phylogenetic analyses
Scutellaria strigillosa (참골무꽃)
Scutellaria indica (골무꽃)
Scutellaria pekinensis var. alpina(수골무꽃)
Isodon inflexsus (산박하)
This is a result of your sequencing data!