Generate a DNA Barcode and Identify Species Is

Generate a DNA Barcode and Identify Species Is there something fishy about what you’re eating? Bio-Rad Biotechnology Explorer Fish DNA Barcoding Kit and DNA Barcoding Sequencing Module

Instructors - Bio-Rad Curriculum and Training Specialists Damon Tighe, damon_tighe@bio-rad. com Sherri Andrews, Ph. D. sherri_andrews@bio-rad. com Leigh Brown, M. A. leigh_brown@bio-rad. com 2 Biotechnology Explorer™ | explorer. bio-rad. com

Workshop Timeline § Introduction § Fish DNA extraction § Gel electrophoresis § DNA visualization with UView. TM § Bioinformatics and species identification § Inquiry Questions 3 Biotechnology Explorer™ | explorer. bio-rad. com

Diversity of Life § It is estimated that there are 10 -100 million species of organisms on Earth § Only about 1. 7 million species have been formally identified § Current limitation to studies of biological diversity - humans are limited in their ability to recognize and recall morphological variation § Few taxonomists can even reliably identify a collection of ~1000 species § How do we complete the task of identifying the remaining species, let alone recognizing them once they are identified? § Solution – Create a genetic based identification system (DNA barcode) 4 Biotechnology Explorer™ | explorer. bio-rad. com

Visual Classification § Some distinct species are not easy to differentiate by eye… vs or 5 Biotechnology Explorer™ | explorer. bio-rad. com

What is DNA Barcoding? § A worldwide effort (International Barcode of Life, i. BOL) exists to “barcode” or generate standard genetic sequence identification of all species on Earth. § What is a barcode? – UPC (Universal Product Code) Symbol – 11 variable positions with 10 possible numbers – Ability to assign a unique identifier to over 100 billion items § What is a DNA barcode? – Use of a designated DNA sequence to serve as a unique species identifier – Ideal sequence is constrained by overall conservation (preserve gene function), but still has substantial sequence variation which differentiates species CCCTCCTA 6 Biotechnology Explorer™ | explorer. bio-rad. com

Barcode Of Life (BOL) § i. BOL – – International Barcode of Life Project Hub is at Biodiversity Institute of Ontario (BIO) at U Guelph Goal to generate 5 million barcodes representing 500, 000 species Currently: 2 million barcodes representing 300, 000 species in the database – Opportunity to contribute to the global initiative to barcode life on Earth! 7 Biotechnology Explorer™ | explorer. bio-rad. com

Be a citizen scientist! § Participate in the largest biodiversity cataloging project ever undertaken and help build a genetic registry of life § Design a market study to look at local food supply, or local flora and fauna Axolotl / Mexican salamander (critically endangered) 8 Biotechnology Explorer™ | explorer. bio-rad. com

“Sushigate” § § § 9 2008, two 11 th graders in New York did a market substitution study Surveyed 60 samples collected from 4 restaurants and 10 grocery stores Of the 60 samples, 54 could be genetically identified 13 of the 54 were mislabeled (23%)! 2/4 restaurants and 6/10 grocery stores had sold mislabeled fish Biotechnology Explorer™ | explorer. bio-rad. com

“Sushigate” § § § 10 7/9 samples listed as Red Snapper were mislabeled, and included substitutions: Acadian redfish from North Atlantic, Pinjalo from SE Asia, Lavender jobfish from So. Pacific, Nile perch from Africa, and Atlantic Cod. Spotted Goatfish (restricted to the Caribbean) sold as Mediterranean Red Mullet White Bass (farmed freshwater fish) sold as Sea Bass Smelt Roe sold as Flying Fish Roe White (albacore) tuna sushi was Mozambique tilapia (commonly farmed) Biotechnology Explorer™ | explorer. bio-rad. com

Food Fraud in the News 11 Biotechnology Explorer™ | explorer. bio-rad. com

DNA Barcode Region Defined Genes Designated as Barcode Regions: § Fungi – ITS – nuclear ribosomal internal transcribed spacer region § Plants – 2 genes required – rbc. L – chloroplast ribulose-1, 5 -bisphosphate carboxylate – mat. K – chloroplast maturase K § Animals – COI – mitochondrial cytochrome C oxidase subunit I § Why COI? – – – 12 Mitochondrial genome lacks introns Limited exposure to recombination Haploid mode of inheritance Universal primers are robust Hundreds to thousands of mitochondria/cell – this means many more copies of the COI gene in your sample! Biotechnology Explorer™ | explorer. bio-rad. com Mitochondrial DNA

Applications of DNA Barcoding What did I eat last night (and is it what they said it was)? What did I catch yesterday? What is the genetic signature of this rare species? 13 Biotechnology Explorer™ | explorer. bio-rad. com

Fish DNA Barcoding Kit Start to Finish DNA BARCODE 14 Biotechnology Explorer™ | explorer. bio-rad. com

DNA Barcoding Kit Workflow PCR amplification Fish sample Extract genomic DNA Sequencing, Sequence Analysis Gel electrophoresis 15 Biotechnology Explorer™ | explorer. bio-rad. com

Barcoding Overview 16 Biotechnology Explorer™ | explorer. bio-rad. com

Barcoding Overview 17 Biotechnology Explorer™ | explorer. bio-rad. com

Barcoding Overview 18 Biotechnology Explorer™ | explorer. bio-rad. com

DNA Extraction Overview + Resuspension + Lysis Buffer Bind DNA to column (Matrix Solution) Wash column with Wash buffer 19 Biotechnology Explorer™ | explorer. bio-rad. com Incubate 10 min at 55 o. C + Neutralization Buffer Elute DNA

Quick Guide – Fish Prep 1 2 20 Label tubes “ 1” for fish sample 1, “ 2” for fish sample 2. Also label with your initials. Cut a piece of fish approximately the size of a pencil eraser-head, from your first fish sample. Slice it until finely minced. Transfer the sample into microcentrifuge tube 1. Biotechnology Explorer™ | explorer. bio-rad. com 1 2 1

Quick Guide – Fish Prep 3 4 21 Using a new cutting implement, cut a piece of fish approximately the size of a pencil eraser-head, from your second fish sample. Slice it until finely minced. Transfer the sample into microcentrifuge tube 2. Add 200 ml of Resuspension to your two tubes and flick several times to ensure full submersion of the fish in the resuspension solution. Biotechnology Explorer™ | explorer. bio-rad. com 2

Quick Guide – DNA Extraction 5 Add 250 µl of Lysis to each tube and mix gently by inverting tubes 10 times to mix contents. 6 Incubate samples at 55 o. C for 10 min. The samples do not need to be shaken during incubation. 22 Biotechnology Explorer™ | explorer. bio-rad. com

What is happening during DNA extraction? Where is the DNA at each step? § Resuspension – buffered solution with chelating agents to destabilize cell membranes – DNA: pellet (fish) or supernatant? § Lysis – alkaline solution that disrupts membranes, releases DNA, denatures DNA – DNA: pellet (fish) or supernatant? § Heating – helps to break down tissue to recover more DNA § Neutralization – solution that counteracts the effects of alkalinity, renatures smaller pieces of DNA, helps precipitate DNA and remove detergents – DNA: pellet (fish) or supernatant? 23 Biotechnology Explorer™ | explorer. bio-rad. com mito DNA nuclear DNA

What is happening during DNA extraction? Where is the DNA at each step? § Matrix – silica based suspension that binds DNA but not RNA or proteins – DNA: column or flow through? pelleted proteins, membranes, etc § Wash – removes other small particles in the prep that are nonspecifically bound to the Matrix – DNA: column or flow through? § Elute – low ionic strength buffer or water releases DNA from the silica – DNA: column or flow through? 24 Biotechnology Explorer™ | explorer. bio-rad. com mito DNA

Quick Guide – DNA Extraction 7 Add 250 ml of Neutralization to each tube and mix gently by inverting tubes 10 times to mix. A visible cloudy precipitate may form. 8 Centrifuge the tubes for 5 min at top speed (14, 000 x g) in the microcentrifuge. A compact pellet will form along the side of the tube. The supernatant contains the DNA. 25 Biotechnology Explorer™ | explorer. bio-rad. com

Quick Guide – DNA Extraction 9 10 26 Snap (do not twist!) the bottoms off of the spin columns and insert each column into a capless 2 ml microcentrifuge tube. Label columns 1 and 2 + your initials. Transfer the entire supernatant (500– 550 µl) of each fish sample into the appropriately labeled spin column. Try not to get any of the particulates into the spin column because they will clog the column and prevent you from continuing. Biotechnology Explorer™ | explorer. bio-rad. com 1 1 2 2

Quick Guide – DNA Extraction Thoroughly mix the tube labeled Matrix to make sure particulates are completely resuspended before use. 11 12 1 Add 200 ml of thoroughly resuspended Matrix to the first column and pipet up and down to mix. Using a new pipet tip, add 200 ml of thoroughly resuspended Matrix to the second column and pipet up and down to mix. 13 Centrifuge the columns for 30 sec at full speed. Remove flow through to waste. 27 Biotechnology Explorer™ | explorer. bio-rad. com 1 2 2

Quick Guide – DNA Extraction 14 Add 500 µl of Wash and wash the samples by centrifugation for 30 sec. Remove flow through to waste. 1 Wash 15 28 Repeat wash step and centrifugation as shown above. Biotechnology Explorer™ | explorer. bio-rad. com 2

Quick Guide – DNA Extraction 16 Centrifuge columns for a full 2 min to remove residual traces of Wash and dry out the samples 17 Remove the spin columns and discard the 2 ml microcentrifuge wash tubes. Place the spin column for each sample into a new capless 2 ml tube. 1 2 1 new capless tube 29 Biotechnology Explorer™ | explorer. bio-rad. com

Quick Guide – DNA Extraction 18 19 30 Using a fresh pipet tip for each sample, add 100 µl of distilled water to each spin column, being careful not to touch the resin. Elute the DNA by centrifuging for 1 min. Label two clean 2 ml microcentrifuge tubes (with caps) Fish 1 and Fish 2 and your initials. Transfer the eluted DNA into the appropriately labeled tube. Biotechnology Explorer™ | explorer. bio-rad. com 1 1 2 2

PCR amplification of COI gene § Fish DNA has been extracted § Next step is to amplify a portion of the mitochondrial COI gene – Generate enough DNA to visualize on a gel – Generate enough DNA to send for sequencing § Assemble reactions of – – – Template DNA Primers Nucleotides Taq polymerase Magnesium chloride § Multiple rounds of thermal cycling to amplify DNA 31 Biotechnology Explorer™ | explorer. bio-rad. com Mitochondrial DNA

PCR – Degenerate primers § § When trying to amplify DNA from a wide variety of samples (many different fish) using the same primer set, creating degenerate primers is a useful approach Determine a consensus sequence derived from several species – – – § § 32 Pike: Carp: Tuna: Bass: Hake: CONS. : A-C-T-G-G-C-T-T-A-G-C A-C-T-G-G-A-T-T-A-G-C A-C-T-G-G-G-T-T-A-A-C A-C-T-G-G-T-T-T-A-G-C A-C-T-G-G-A-T-T-T-A-C A-C-T-G-G-N-T-T-A-R-C The consensus/degenerate primers bind to DNA from all of these fish, whereas regular primers would only bind to one The primers used in our Fish DNA barcoding kit contain degenerate positions to amplify DNA from as many different fish as possible! Biotechnology Explorer™ | explorer. bio-rad. com

Fish Barcoding PCR Primers 33 Biotechnology Explorer™ | explorer. bio-rad. com

PCR – Overview Heat (94 o. C) to denature DNA strands Cool (55 o. C) to anneal primers to template Warm (72 o. C) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles Your PCR products will be given to you now for electrophoresis 34 Biotechnology Explorer™ | explorer. bio-rad. com

Quick Guide - Electrophoresis Add 2 µl of UView 6 x loading dye to each sample, using a new pipet tip each time. Mix samples well. Load the agarose gel in the following lane order and volumes, using a new pipet tip each time: Lane 1 2 3 4 5 6 7 8 35 Sample EMPTY 20 µl MWR 12 µl (+) E 12 µl (–) E 12 µl 1 E 12 µl 2 E EMPTY Biotechnology Explorer™ | explorer. bio-rad. com 200 V 20 min 0. 25 x TAE

Visualizing DNA after electrophoresis UView. TM Nontoxic Loading dye + Stain Instant View with UV Very sensitive 36 Ethidium Bromide Toxic, Mutagen Stain Instant View with UV Most sensitive Biotechnology Explorer™ | explorer. bio-rad. com Fast Blast. TM Nontoxic Stain Requires wait time View by eye Less sensitive

Use UV transilluminator to visualize UView or Ethidium Bromide 1. 2. 3. 4. 5. MW ruler (+) control (-) control Fish 1 Fish 2 UView. TM Ethidium bromide 1 37 Biotechnology Explorer™ | explorer. bio-rad. com 2 3 4 5

Sequencing of PCR products 38 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Alignment Run . . . What is the longest run of tails I should expect for 100 tosses? R = log 1/p (n) Paul Erdos‐Alfréd Rényi law R = longest run p = probability (for “fair” coins its 0. 5) n = number of tosses Paul Erdos 39 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Alignment Run . . . What is the longest run of tails I should expect for 100 tosses? R = log 1/0. 5 (100) = 6. 64 Paul Erdos 40 …. so if I get more than 6. 64 tails in a row when tossing 100 times, I might wonder if something besides randomness is going on Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Alignment AATCGTACTG AACCATTCAG If I call alignments tails. What is the longest run of tails I should expect for comparing two 10 bp sequences? R = log 1/0. 25 (100) = 3. 32 Sequence lengths multiplied ¼ chance for getting same base 41 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Alignment DNA is not a 4 sided Coin - Account for probability of bases being switched out for each other by a scoring matrix Match Transversion Change of base type – Purine for Pyrimidine 42 Biotechnology Explorer™ | explorer. bio-rad. com Transition Same base type – Purine for Purine

Bioinformatics – Alignment DNA is not a 4 sided Coin 43 G A A T T C A G T T A G 1 1 1 G 1 1 1 1 2 2 A 1 2 2 2 2 2 T 1 2 2 3 3 3 3 C 1 2 2 3 3 4 4 4 G 1 2 2 3 3 4 4 5 5 A 1 2 3 3 3 4 5 5 6 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Alignment DNA is not a 4 sided Coin 44 G A A T T C A G T T A G 1 1 1 G 1 1 1 1 2 2 A 1 2 2 2 2 2 T 1 2 2 3 3 3 3 C 1 2 2 3 3 4 4 4 G 1 2 2 3 3 4 4 5 5 A 1 2 3 3 3 4 5 5 5 Biotechnology Explorer™ | explorer. bio-rad. com 5 5 6

Bioinformatics – BLAST tool Query Database ATTCGCAT ATT TTC TCG CGC GCA CAT 1) Break into words 2) Find Matches and let go of all the other database words that don’t match 3) Extend from match 1 base at a time until score falls off 4) Use two anchors to define and alignment, compare, score E-value 45 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – BLAST tool E-value Theoretically, we could trust any result with an E-value ≤ 1 In practice – BLAST uses estimations. • E-values of 10 -4 and lower indicate a significant homology. • E-values between 10 -4 and 10 -2 should be checked (similar domains, maybe non-homologous). • E-values between 10 -2 and 1 do not indicate a good homology 46 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics – Linking DNA Barcoding and Protein Profiler + Compare light chain of myosin sizes = Compare E-value for CO 1 gene Stronger Evidence for Evolutionary Relationship 47 Biotechnology Explorer™ | explorer. bio-rad. com

Bioinformatics using BOLD-SDP = Barcode Of Life Data systems – Student Data Portal Quick Start tutorial available online 48 Biotechnology Explorer™ | explorer. bio-rad. com

Create an Instructor Account 49 Biotechnology Explorer™ | explorer. bio-rad. com

Fill Out Required Information 50 Biotechnology Explorer™ | explorer. bio-rad. com

Receive Two Important Emails – Login and Registration Keys Registration keys 51 Biotechnology Explorer™ | explorer. bio-rad. com

Log In and Register a New Course 52 Biotechnology Explorer™ | explorer. bio-rad. com

Fill in Course Info, List Students, Receive Class Login 53 Biotechnology Explorer™ | explorer. bio-rad. com

Students Log In and Enter Specimen Info 54 Biotechnology Explorer™ | explorer. bio-rad. com

Enter Specimen Information 55 Biotechnology Explorer™ | explorer. bio-rad. com

View Class Specimen List 56 Biotechnology Explorer™ | explorer. bio-rad. com

Upload Sequencing Data Files 57 Biotechnology Explorer™ | explorer. bio-rad. com

Select PCR and Sequencing Primers Used 58 Biotechnology Explorer™ | explorer. bio-rad. com

Class List Updates with Specimen Records Uploaded 59 Biotechnology Explorer™ | explorer. bio-rad. com

View Data 60 Biotechnology Explorer™ | explorer. bio-rad. com

Uploaded trace files receive data quality assessment 61 Biotechnology Explorer™ | explorer. bio-rad. com

Trace files viewable 62 Biotechnology Explorer™ | explorer. bio-rad. com

Quality Scores Light blue bars in background represent assigned quality value for each nucleotide (scale on right axis) High quality values Examine peaks Low quality value Examine peaks (mixed call – overlap) 63 Biotechnology Explorer™ | explorer. bio-rad. com

Generate contig (“sequence”) from trace files 64 Biotechnology Explorer™ | explorer. bio-rad. com

Examine base calls in contig Contig sequence 65 Biotechnology Explorer™ | explorer. bio-rad. com

Contig generated, trim primer sequences 66 Biotechnology Explorer™ | explorer. bio-rad. com

Run contaminant check and submit contig Contig 573 bp with no ambiguous nucleotides 67 Biotechnology Explorer™ | explorer. bio-rad. com

Data summary barcode generated from data contig sequence translation 68 Biotechnology Explorer™ | explorer. bio-rad. com

Search full database for genetic match 69 Biotechnology Explorer™ | explorer. bio-rad. com

Search species database for genetic match 70 Biotechnology Explorer™ | explorer. bio-rad. com

Species match! 71 Biotechnology Explorer™ | explorer. bio-rad. com

Education and DNA Barcoding - Resources § § § 72 www. educationandbarcoding. org Links to content to aid in classroom presentations Check out ongoing student barcoding campaigns Register your own barcoding campaign! Link to BOLD-SDP workbench (Student Data Portal) Engage in citizen science Biotechnology Explorer™ | explorer. bio-rad. com

Student Inquiry Questions to consider: – How important is each step in the lab protocol? – What part of the protocol can I manipulate to see a change in the results? – Possible variables / questions: • How will results be affected by the use of different fish sources (fresh, frozen, dried, canned)? • Will different fish tissue yield better results (muscle vs fin, gills, or scales)? • Cleanliness and attention to detail during fish processing – How do I ensure the changes I make are what actually affects the outcome (importance of controls). – Write the protocol. After approval – do it! 73 Biotechnology Explorer™ | explorer. bio-rad. com

Student Inquiry - Teacher Considerations § What materials and equipment do I have on hand, and what will I need to order? – Extra gels, different organisms? – Other supplies depending on student questions – Consider buying extras in bulk or as refills – many have 1 year + shelf life. § What additional prep work will I need? – Order supplies 74 Biotechnology Explorer™ | explorer. bio-rad. com

Student Inquiry - Teacher Considerations 75 Biotechnology Explorer™ | explorer. bio-rad. com