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9 Faculty of Science
15 Research Management & Innovation Complex (IPPP)
17 Institute of Graduate Studies (IPS)

DNA barcoding in hyper-diverse Malaysia


22-23 October 2013
University of Malaya, Kuala Lumpur
Tuesday 22nd October
8:30 13:00 Laboratory Workshop: Basics of DNA Barcoding Part 1
Venue: Institute of Graduate Studies (IPS)
(Pages 6)
2:30 4:00 Public Seminar: The International Barcode of Life
Venue: Research Management & Innovation Complex (IPPP)
(Page 4)
Wednesday 23rd October
10:30 - 12:30 Scientific Meeting: Activation of Malaysia as an iBOL partner
Venue: Research Management & Innovation Complex (IPPP)
(Page 5)
2:30 4:30 Laboratory Workshop: Basics of DNA Barcoding Part 2
Venue: Faculty of Science (Block C)
(Page 7)

Organiser:
Dr. John James Wilson

Planning Committee:
Prof. Mohd Sofian-Azirun

Institute of Biological Sciences, UM

Dean, Faculty of Science, UM


Head, Zoological and Ecological Research Network

Secretary:
Dr. Evan Chin Sui See
Lab Assistants:
Mr. Gary Sing Kong Wah
Miss. Kharunnisa Syarippudin
Miss. Lee Ping Shin

Prof. Rosli Hashim


Head, Institute of Biological Sciences, UM

Dr. A. Sasekumar
Curator, Museum of Zoology, UM

Dr. Chen Chee Dhang


Research Officer, ZEN

Supported by:

iBOL Partner Nations: Case Studies

Comprises: Universities and research institutes in Mexico (Institute of Biology, UNAM, South Frontier
College, ECOSUR, and Center for Biological Research of the Northwest, CIBNOR).

Funding: The Government of Mexico through CONACYT, the National Council on Science and
Technology, and CONABIO, the National Commission for Knowledge and Use of Biodiversity.

Outputs: Hosted the International Barcode of Life meeting in 2009. Published a special issue of
Mitcohondrial DNA (IF=1.705) in 2010. Other research papers in PLoS ONE, Molecular Ecology Resources.

Comprises: Seoul National University and


other university researchers.

Funding: The Ministry of Environment and


the Ministry of Education, Science and
Technology of Korea .

Outputs: Korea Barcode of Life database


system (KBOL) and associated publications (BMC
Genomics, Animal Cells and Systems).

Pakistan iBOL node


Comprises: National Institute for
Biotechnology and Genetic Engineering (NIBGE).

Funding: Higher Education Commission


Pakistan.

Outputs: Large amount of sequence data

KENBOL - the Kenyan Barcode


of Life Project
Comprises: National Museums of Kenya
(NMK), Kenya Marine and Fisheries Research
Institute (KMFRI), Kenya Wildlife Service (KWS)
and icipe -African Insect Science for Food and
Health.

Funding: International Development


Research Centre (IDRC), Canada.

Outputs: None yet but early days.

assembled. Publication in Molecular Ecology


Resources.

Public Seminar:
The International Barcode of Life
22nd October 2:30pm
Research Management & Innovation Complex (IPPP) Auditorium,
University of Malaya
by

Paul D. N. Hebert,
University of Malaya Visiting Professor
Dr. Paul D. N. Hebert is the founder and Scientific Director of the International
Barcode of Life (ibol.org) and Director of the Biodiversity Institute of Ontario
and the Canadian Barcode of Life Network. He holds a Canada Research Chair
in Molecular Biodiversity at the University of Guelph, has published more than
300 papers (including Nature), has received national and international
scientific awards and is a Fellow of the Royal Society of Canada. His 2003
research paper proposing a database of DNA barcodes to enable the
identification of all species has received more than 3300 citations.
Contact:
Paul D. N. Hebert
phebert@uoguelph.ca
Biodiversity Institute of Ontario
University of Guelph,
Guelph, Ontario, N1G 2W1,
Canada

Basics of DNA Barcoding Workshop Part 1


These instructions have been modified from:
Wilson JJ (2012) DNA barcodes for insects. In: Kress WJ
and Erickson DL (Eds.) DNA barcodes: Methods and
protocols: Methods in molecular biology (pp. 17-46).
doi:10.1007/978-1-61779-591-6

Name:
Sample Number:

A) Tissue Sampling
1. Take the forceps and dip them in ethanol (carefully shaking off any excess, not near the flame)
and put them in the flame for a few seconds to burn off the ethanol.

2. Working over a KimWipe, remove a small piece of tissue from the specimen (about a 2-3mm-long
piece of insect leg) and place it in the tube provided (labelled gDNA).

B) DNA extraction
(using Xytogen ANDE kit; http://dx.doi.org/10.1016/j.aspen.2010.04.003)
1. Add 5.5uL of Solution 2 (labelled S2). Seal tube tightly. Make sure the tissue is fully submerged in
the liquid.

2. Incubate at 95C in the PCR machine for 20min.

3. Add 6.25uL of Solution 3 (labelled S3). Seal tube tightly and invert tube gently 3 times.
C) PCR set up

(using Econotaq PLUS GREEN mastermix; http://lucigen.com)

1. Add 1uL of gDNA extract to each tube containing PCR master mix (labelled PCR1 and PCR2).
PCR1 contains primers LepF and LepR. PCR2 contains primers MLepF and LepR.
2. Centrifuge the tube at 1000xg for 20s and place in the PCR machine.

COI fast PCR program: 94C for 1min; five cycles of 94C for 30s, annealing at 45C for 40s, and
extension at 72C for 1min; followed by 35 cycles of 94C for 30s, annealing at 51C for 40s, and
extension at 72C for 1min; with a final extension at 72C for 10mins.
D) PCR check
1. Retrieve your sample from the PCR machine and centrifuge the tube at 1000xg for 20s.

A 1% agarose gel has been prepared using 1XTAE buffer.


2. With a steady hand load 3uL of each of your PCR products into the assigned wells on the gel
3. Pass the tube to be sent for sequencing.

A 150V current is applied to the gel for 20mins to electrophoretically separate the DNA fragments.
DNA is negatively charged and will move towards the positive electrodes. After this time, the gel is
removed from the rig and stained with DNA sensitive dye which fluoresces under UV light to
enable visualization of PCR success.
We will visualize the PCR products and send the successful reactions for sequencing overnight
with local company. In Part 2 you will find out if your PCR was successful and the next steps.
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Basics of DNA Barcoding Workshop Part 2


E) Sequence editing

(using CodonCode Aligner)

1. The sequences come back from the sequencer by email as a .zip file. Each sample comes back in
two formats, .ab1 file and .txt file. The files you are interested in have extension .ab1. Create a
folder on your desktop called Traces and place all the .ab1 files into the folder.
2. Open CodonCode and choose Create a new project and press OK. Go to File>Import>Add
Folder>Traces then press Import.
3. To see the files you just imported press besides the Unassembled Samples folder.
4. Sort files by quality by double clicking on Quality. Any sequences that are of very poor quality, or
of short length, highlight them and click the trashcan to delete them.
5. Next select the Contig menu and move the cursor over Advanced Assembly. From the options that
appear select Assemble in Groups. A window will appear asking if you would like to Define
sample name parts? Choose Define names... to bring up another small window.
6. There are two parts to our sample names (e.g. AA_F). The first part is the sample number (e.g.
AA) and the option in the Meaning menu can be left as Clone. Since the sample number is
followed by an underscore, choose _ (underscore) in the Delimiter menu for Clone.
7. The second part is the direction (e.g. F) so choose Direction in the Meaning menu. We can
ignore the Delimiter for Direction part.
8. Click Preview... to check how Aligner is interpreting the sample names. Click Close to exit the
preview. Click OK to return to return to the Assemble in Groups window.
9. We want to assemble our files according to direction. Choose Direction in the Name Part section.
Then click Assemble. You should now have two folders (two contigs), one called F with the
forward sequences and one called R with the reverse sequences.
10. Next you need to cut the primers from your sequences.
11. Highlight the R folder and reverse and complement the sequences using the button with three
black arrows on it. Double click the R folder to open it. For the reverse sequences you need to
find the forward primer motif (e.g. LepF) and delete it from the beginning of the consensus
sequence at the bottom of the window. You will find the primer around 50 nucleotides from the
end of the raw sequence. For example, you would need to delete the section of the sequence
marked below in bold and everything to the left of it. Highlight it on the consensus sequence
and press the Backspace key on the keyboard.
ATTCAACCAATCATAAAGATATTGGAACATTATATTTTATTTTTGGTATTTGATCAGG.....
12. Next go to the opposite end of the consensus, the far right. Delete the consensus sequence from
the point where the sequences get messy. This will show in green highlight, and probably with
the present of gaps (-). For example, delete the section marked in bold and everything to the

right of it. Highlight it on the consensus sequence and press the Delete key on the keyboard.
Close the window.
...TCTTTTTTTGACCCTGCTGGTGGAGG-G-TTT-GGT-AA-T-TTT-G-C
13. Double click the F folder to open it. Go to the far right of the consensus sequence and find the
reverse and complement of the reverse primer motif (e.g. LepR) at the very end. This should be
around 690-700 bp on the raw sequence. For example, you would delete the section marked in
bold and everything to the right of it. Highlight it on the consensus sequence at the bottom of
the window and press the Delete key.
...GGGGAGACCCTATTCTTTATCAACATTTATTTTGATTTTTTGGACATCCAGAACTTTA
14. Next go to the opposite end of the consensus, the far left, and delete the consensus sequence
from the point where the sequences get messy. This will show in green highlight, and probably
with the presence of gaps (-). For example, you would delete the sequence in bold and and
everything to the left of it. Highlight the region on the consensus sequence and press the
Backspace key on the keyboard. Close the window.
AT-GC-T-TTTT-TTT-G-A-TGTTTA-ATCAGGACTAATTGGAACTTC
15. Dissolve both the F and R folders by selecting them and clicking on the button marked X.
16. Select all the sequences and press the button marked with N. This time in order to assemble our
sequences by sample (i.e. one forward and one reverse) choose Clone in the Name Part menu,
then click Assemble. Note, specimens which only sequenced successfully in one direction will
have files which remain in the Unassembled Samples folder.
17. Open each folder (contig) in turn by double-clicking. Correct ambiguous positions (shown in red
e.g. TAT) and gaps (-) in the consensus sequence by checking the original traces. This is done
by double-clicking on the consensus sequence. Always check both trace files (forward and
reverse) and compare them. Note, the corrected consensus sequence should have NO gaps.
18. Generally if reads conflict, you can decide which is more reliable based on sequence quality (e.g.
less background noise, taller peaks).
19. Check the contigs first, then check the single sequences in the Unassembled samples folder.
20. To export the consensus sequences select all the folders using shift click, go
File>Export>Consensus sequences...., choose Current selection. Press Export. Save the file to the
desktop as sequences.fas.

F) Sequence identification
Open sequences.fas in MS Word. Copy the sequence and paste into:

http://boldsystems.org/index.php/IDS_OpenIdEngine
http://blast.ncbi.nlm.nih.gov/Blast.cgi
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