DNA sequencing: Difference between revisions
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== Overview ==
DNA barcoding enables the identification of unknown organisms, discovery of new species, the determination of which features delineate species, accurate determination of the range of a species and the building of phylogenetic trees which show how various organisms are related. The most powerful thing that DNA barcoding does is it tells you which collections are the same species. If your goal is to get insights into strains of the same species or learn which genes are present, you'll need to use a different process called full genome sequencing.
Step by step photos of the whole process are available here: https://www.facebook.com/groups/FungalSequencing/permalink/2268745516680176/
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A video which shows the whole DNA barcoding process is here: https://www.facebook.com/groups/FungalSequencing/permalink/2276807085874019/
Sigrid's PCR protocol:
How to upload DNA sequences to Genbank: https://www.facebook.com/groups/FungalSequencing/permalink/2163215977233131/
Jerry Cooper's excellent Notes on home-based PCR for barcoding fungi: https://www.funnz.org.nz/sites/default/files/DIY%20DNA%20PCR_2.pdf
Nanopore DNA sequencing protocol by Stephen Russell: https://www.protocols.io/view/ont-dna-barcoding-fungal-amplicons-w-minion-amp-fl-36wgq7qykvk5/v2/protocols
If you would like to send in a mushroom and pay about $25 to have it barcoded, you can send to to http://alvalab.es in Spain.
You can also send mushrooms to the Ohio Mushroom DNA Lab for sequencing. They accept donations but do not charge a fee. https://docs.google.com/document/d/1nSKlouFy1Z3OXbCf2cWiKH0hkDnFKZvPn6_oCr3vEK0/edit
== DNA Extraction ==
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The goal of DNA extraction is to get a few nanograms of your organism's DNA into solution so it can be used as a [https://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR template].
Two good DNA extraction methods are the NaOH extraction and [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/extract-n-amp.html ExtractNAmp]. In a pinch you can just use water.
* NaOH extraction (Wang et al. 1993, Osmundson et al. 2013). 200 μL of 0.5 M NaOH is added to the ground tissue. 5 μL of the extract is diluted in 495 μL of 100 mM Tris-HCl, pH 8.0 (pH is adjusted with pH meter and HCl until it is 8.0), and 1 μL of the dilution is used as template DNA for a 25 μL PCR reaction.
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The amount of mushroom you use is not critical, but smaller pieces often have a higher chance of working since there is less likelihood of contamination, and smaller pieces of mushroom allow you to use less DNA extraction reagent. I usually use about 20 milligrams, and if it is a tiny collection and I want to use less I use less DNA extraction solution. Since only 1 uL is needed for the template DNA, in theory you can get a good sequence from a piece of mushroom that is nearly too small to see. If the mushroom has gills, use that part because they have more DNA. If the mushroom is thick, break it in half and use part of the inside of the cap to reduce the chance of contamination. Get the mushroom piece with forceps and heat sterilize them between samples to reduce cross contamination. Flat tipped forceps may not need heat sterilization, you can just wipe them off. Sometimes I grind the samples with plastic pestles after adding extraction solution, but this isn't necessary and I am not sure if it helps. Grinding the samples probably increases the chance of success with something hard like a polypore but decreases the chance of success if your sample includes PCR inhibitors.
I suggest doing DNA extractions in .2 ml PCR tubes - that way you can incubate the DNA extractions in your thermocycler, and they are easier to store in the freezer. It is best to save DNA extractions so you can rerun PCR later with different primers to sequence additional genes.
PCR works at a wide range of DNA concentrations, and a likely cause of PCR failure is the presence of PCR inhibitors. More lengthy DNA extraction protocols can separate the DNA from PCR inhibitors. This is more often required with samples that have a lot of pigments.
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Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples https://nature.berkeley.edu/garbelotto/downloads/naohextraction2013.pdf
In a pinch, plain water can be used as DNA extraction solution.
== Polymerase Chain Reaction (PCR) ==
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I use a 25 μL [https://en.wikipedia.org/wiki/Polymerase_chain_reaction PCR] reaction which includes 1 μL of 10 μM forward [https://en.wikipedia.org/wiki/Primer_(molecular_biology) primer], 1 μL of 10 μM reverse primer, 1 μL DNA template and the [https://www.google.com/search?q=pcr+master+mix&oq=PCR+master+mix PCR master mix]. I use the Taq
The current PCR program I am using is an initial denaturation of two minutes at 95 degrees C, followed by 30 cycles of denaturation at 95 degrees for 30 seconds, annealing at 54 degrees for 30 seconds and an extension phase of 72 degrees for 55 seconds. Since the DNA we are amplifying typically isn't very long, it is probably ok to omit the final extension phase of 6 minutes at 70 degrees. The 54 degree annealing temperature was chosen by looking up the [http://biotools.nubic.northwestern.edu/OligoCalc.html melting point] of the primers and subtracting 3 - 5 degrees.
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==Gel Electrophoresis==
Electrophoresis can tell you if your PCR worked. You can skip this step if you want to save time - you will spend a little more on sequencing PCR reactions that did not work.
[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis]
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If the gel electrophoresis indicates that the PCR reaction worked, it can be brought in for sequencing. For sequencing I use [http://www.genewiz.com/ Genewiz], which will pick up samples directly from CCL. If you fill out the sequencing order by 3:30pm, the courier arrives around five and you will get the sequences at about 6am the next morning. If you sign up for a new account, the first two sequences are free. If you have Genewiz do the DNA quantification and PCR cleanup, they charge $7 per sample or $5 per sample if you do 96 at a time.
[
Lower cost sequencing options are available, though you need to mail in the PCR products rather than having them picked up if you aren't near the lab. Two low cost Sanger sequencing options are [https://www.eurofinsgenomics.com/en/products/dna-sequencing/all-sequencing-options/ Eurofins] and [http://www.mclab.com/DNA-Sequencing-Services.html McLab]. Mclab is the least expensive, at $5 per sample for a bidirectional read with PCR cleanup (if you order 8 reactions at a time), which is less than half of what Genewiz charges. Often the forward works and the reverse doesn't, or vice versa - usually one of them works unless you have multiple templates being amplified or some other problem. Mclab can be a little tricky to order from because they want you to fill out an Excel order form - it's not difficult if you have a template to work from, available [https://images.mushroomobserver.org/Mclab%20DNA%20barcoding%20sample%20form.xlsx here.]
Another way to sequence PCR products is with a MinION DNA sequencer. This method is much newer than Sanger and has much higher startup cost, but once it is running it is a whole lot faster and less expensive than Sanger sequencing - with similar quality data. For more information on how to do this, see https://www.protocols.io/view/ont-dna-barcoding-fungal-amplicons-w-minion-amp-fl-36wgq7qykvk5/v2/protocols.
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==Supplies Needed==
* PCR machine [https://www.ebay.com/sch/i.html?_nkw=PCR+machine $349] (avoid the cheap Thermo Hybaid brand, Josiah says these break really easily. Good ones are Biorad, MJ Research
* Gel box with tray and comb [https://www.ebay.com/sch/i.html?_nkw=Gel+Electrophoresis about $100] or [http://citizensciencequarterly.com/2011/10/cheapass-science-gel-box/ make your own for $21]▼
▲* PCR machine [https://www.ebay.com/sch/i.html?_nkw=PCR+machine $349] (avoid the cheap Thermo Hybaid brand, Josiah says these break really easily. Good ones are MJ Research (usually about $400) and Applied Biosystems (reliable and cheap, like $100, but huge - shipping is like $75)
* 10 uL and 200 uL pipettes [http://www.ebay.com/itm/NEW-Oxford-10-50uL-Adjustable-Micro-Pipette-Series-3000-Sampler-System-Pipet-/291224844297?hash=item43ce5a7809:g:lQIAAOSwxCxT96LG 10 - 50 uL $20] [http://www.ebay.com/itm/Brand-New-Single-Channel-Adjustable-Pipetman-20-200uL-p200-pipet-Pipette-j2e-/182032059910?hash=item2a61f4ce06:g:40QAAOSwiylW~eU2 P200 $22]
* PCR master mix [https://www.the-odin.com/taq-polymerase-master-mix-2x-1ml-40-reactions/ $30 for 40 reactions] [https://empiricalbioscience.com/shop/rae/grouping-2-rae/taq-2x-colorless-mastermix-4/ $88 for 500 reactions]
* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50]▼
* Pipette tips (sterile, for p10 and p200 pipettes) $40
* PCR tubes [http://www.ebay.com/itm/Azzota-PCR-Tubes-Flat-8-strip-Caps-120pcs-pk-/252184303378?hash=item3ab75b1712:g:9DUAAOSwt6ZWVJCA $10 for 120 tubes]
* DNA extraction supplies (or IBI X-Amp) (NaOH and TRIS buffer, optionally plastic pestles. If you get pestles get the size for the .5 ml tubes so you can use 200 uL PCR tubes for DNA extractions) [https://www.ebay.com/sch/i.html?_nkw=Sodium+Hydroxide $10] [https://www.ebay.com/sch/i.html?_nkw=TRIS+base $10]
* Ethidium Bromide or other DNA stain [http://www.the-odin.com/gel-green-like-dna-stain-for-gels-10-000x-100ul/ $10]▼
*
* Agarose [http://www.the-odin.com/agarose/ $10]▼
* Gel loading dye [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5]▼
* TAE buffer concentrate [http://www.the-odin.com/tae-buffer-mix-10-l-50g/ $10]
* Tube rack for DNA extracts [http://www.the-odin.com/microcentrifuge-tube-rack-60-tube-2-sided-1-5-ml-and-0-5-or-0-2-ml-reversible/ $5]
* Pure, DNA-free water $2 (bottled distilled water works)
▲* Gel box with tray and comb [https://www.ebay.com/sch/i.html?_nkw=Gel+Electrophoresis about $100] or [
▲* Electrophoresis DC power supply [https://www.ebay.com/sch/i.html?_nkw=Electrophoresis+power+supply $50] (optional)
▲*
* 100 bp DNA Marker (optional) [http://www.the-odin.com/dna-ladder-100bp/ $10]
▲* Gel loading dye (not necessary if you get master mix that already has loading dye) [http://www.the-odin.com/6x-dual-color-dna-loading-dye/ $5] (optional)
* Blue LED light (490 nanometer) and orange glasses for viewing DNA on gel (a 365 nanometer UV flashlight works even better - no orange glasses needed) [https://www.amazon.com/dp/B005IPPBNI $17] (optional)
▲* Agarose [http://www.the-odin.com/agarose/ $10] (optional)
* Total cost: $895 (or about $200 less if you DIY some of the pieces)
== Location of equipment in CCL ==
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