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

Workshop Timeline Introduction Fish DNA extraction Gel electrophoresis DNA visualization with UViewTM Bioinformatics and species identification Inquiry Questions

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)

Visual Classification Some distinct species are not easy to differentiate by eye… vs or

What is DNA Barcoding? A worldwide effort (International Barcode of Life, iBOL) 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

Barcode Of Life (BOL) iBOL 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!

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)

“Sushigate” 2008, two 11th 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

“Sushigate” 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)

Food Fraud in the News

DNA Barcode Region Defined Genes Designated as Barcode Regions: Fungi ITS – nuclear ribosomal internal transcribed spacer region Plants – 2 genes required rbcL – chloroplast ribulose-1,5-bisphosphate carboxylate matK – chloroplast maturase K Animals COI – mitochondrial cytochrome C oxidase subunit I Why COI? 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! Mitochondrial DNA

Applications of DNA Barcoding What did I eat last night (and is it what they said it was)? What is the genetic signature of this rare species? What did I catch yesterday?

Fish DNA Barcoding Kit Start to Finish DNA BARCODE

DNA Barcoding Kit Workflow PCR amplification Fish sample Extract genomic DNA Sequencing, Sequence Analysis Gel electrophoresis

Barcoding Overview

Barcoding Overview

Barcoding Overview

DNA Extraction Overview Incubate 10 min at 55oC + Resuspension + Lysis Buffer Buffer Bind DNA to column (Matrix Solution) + Neutralization Buffer Wash column with Wash buffer Elute DNA

Quick Guide – Fish Prep Label tubes “1” for fish sample 1, “2” for fish sample 2. Also label with your initials. 1 2 1 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. 2 1

Quick Guide – Fish Prep 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. 3 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. 4

Quick Guide – DNA Extraction Add 250 µl of Lysis to each tube and mix gently by inverting tubes 10 times to mix contents. 5 Incubate samples at 55oC for 10 min. The samples do not need to be shaken during incubation. 6

What is happening during DNA extraction? Where is the DNA at each step? mito DNA 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 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 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? Wash removes other small particles in the prep that are nonspecifically bound to the Matrix Elute low ionic strength buffer or water releases DNA from the silica mito DNA pelleted proteins, membranes, etc

Quick Guide – DNA Extraction Add 250 ml of Neutralization to each tube and mix gently by inverting tubes 10 times to mix. A visible cloudy precipitate may form. 7 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. 8

Quick Guide – DNA Extraction 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. 9 1 2 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. 10 1 2

Quick Guide – DNA Extraction Thoroughly mix the tube labeled Matrix to make sure particulates are completely resuspended before use. 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. 1 2 11 1 2 12 Centrifuge the columns for 30 sec at full speed. Remove flow through to waste. 13

Quick Guide – DNA Extraction Wash 1 2 Add 500 µl of Wash and wash the samples by centrifugation for 30 sec. Remove flow through to waste. 14 15 Repeat wash step and centrifugation as shown above.

Quick Guide – DNA Extraction 1 2 Centrifuge columns for a full 2 min to remove residual traces of Wash and dry out the samples 16 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. 17 1 new capless tube

Quick Guide – DNA Extraction 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. 18 1 2 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. 1 2 19

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 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 Pike: A-C-T-G-G-C-T-T-A-G-C Carp: A-C-T-G-G-A-T-T-A-G-C Tuna: A-C-T-G-G-G-T-T-A-A-C Bass: A-C-T-G-G-T-T-T-A-G-C Hake: A-C-T-G-G-A-T-T-T-A-C CONS.: 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!

Fish Barcoding PCR Primers

PCR – Overview Heat (94oC) to denature DNA strands Cool (55oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles Your PCR products will be given to you now for electrophoresis

Quick Guide - Electrophoresis Add 2 µl of UView 6x 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 Sample 1 EMPTY 2 EMPTY 3 20 µl MWR 4 12 µl (+) E 5 12 µl (–) E 6 12 µl 1 E 7 12 µl 2 E 8 EMPTY 200 V 20 min 0.25x TAE

Visualizing DNA after electrophoresis UViewTM Ethidium Bromide Fast BlastTM Nontoxic Toxic, Mutagen Nontoxic Loading dye + Stain Stain Stain Instant Instant Requires wait time View with UV View with UV View by eye Very sensitive Most sensitive Less sensitive

Use UV transilluminator to visualize UView or Ethidium Bromide 1. MW ruler 2. (+) control 3. (-) control 4. Fish 1 5. Fish 2 UViewTM Ethidium bromide 1 2 3 4 5

Sequencing of PCR products

Bioinformatics – Alignment Run . . . . . What is the longest run of tails I should expect for 100 tosses? R = log1/p (n) WARNING: THIS is a BIOLOGIST hand waiving of this more complex mathematical model….but we need to at least know at some level what the computer scientist are doing, so we can communicate and solve bigger problems. When we align DNA sequences we are in general asking how many times do the sequences have the same bases in a run (streak). To talk about the statistics of runs its easier to start with the classic coin toss problem – How many runs of the same (heads/tails) would I expect if I flipped a coin 100 times? Paul Erdos - Mathematical bad ass and most prolific mathematician ever….he lived in your lifetime!!!, do you know your Erdos number? Paul and his house host Alfred Renyi went after this question and they found a mathematical model that could work to predict runs for coin tosses in 1959. There is a great radio lab piece on this. Paul Erdos‐Alfréd Rényi law R = longest run p = probability (for “fair” coins its 0.5) n = number of tosses Paul Erdos

Bioinformatics – Alignment Run . . . . . What is the longest run of tails I should expect for 100 tosses? R = log1/0.5 (100) = 6.64 Erods and Renyi find out that if you toss a coin 100 times you should expect to see runs of almost 7 heads or tails. Its no opening scene to Rosencrantz and Guildenstern, but its more then most people would guess and gives us a quantitative way to talk about the appearance of runs. ….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 Paul Erdos

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? Biologists found that if you were comparing sequences of DNA you could equate places in a sequence that were the same with the idea of a run, so in the sequence the first two positions are the same (run of 2). because you are comparing two strings (sequences) you have to multiply them together to get the number of “tosses”. Also you have to change the model a little bit now to deal with the new probability of having 4 things your space could be occupied instead of just two. This model gives us a good baseline, but needs further refinement because nucleotides don’t switch out perfectly….so we have to adjust how we calculate the probability R = log1/0.25 (100) = 3.32 Sequence lengths multiplied ¼ chance for getting same base

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 Transition Change of base type – Purine for Pyrimidine Same base type – Purine for Purine

Bioinformatics – Alignment DNA is not a 4 sided Coin 6 5 4 3 2 1 A G C T

Bioinformatics – Alignment DNA is not a 4 sided Coin 6 5 4 3 2 1 A G C T

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

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

Bioinformatics – Linking DNA Barcoding and Protein Profiler + Compare light chain of myosin sizes Compare E-value for CO1 gene = Stronger Evidence for Evolutionary Relationship

Bioinformatics using BOLD-SDP BOLD-SDP = Barcode Of Life Data systems – Student Data Portal Quick Start tutorial available online

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Upload Sequencing Data Files

Select PCR and Sequencing Primers Used

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Uploaded trace files receive data quality assessment

Trace files viewable

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)

Generate contig (“sequence”) from trace files

Examine base calls in contig Contig sequence

Contig generated, trim primer sequences

Run contaminant check and submit contig Contig 573 bp with no ambiguous nucleotides

Data summary barcode generated from data contig sequence translation

Search full database for genetic match

Search species database for genetic match

Species match!

Education and DNA Barcoding - Resources 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

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!

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

Student Inquiry - Teacher Considerations