TITLE: DNA Barcoding in a Field Setting
AUTHORS: Aaron Pomerantz, Mrinalini Watsa, Stefan Prost, Gideon Erkenswick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol was used in the 2018 Genomics in the Jungle field course held at the Inkaterra Green Lab in the Madre de Dios Department of Peru. We were able to use it to take 112 amplicons and multiplex them using a 96 barcode kit on an Oxford Nanopore Technologies MinION sequencer. This protocol can be used to multiplex much larger numbers of samples onto a single flowcell if the amplicons are from differing taxonomic groups. </div><div class = "text-block">We multiplexed the following taxonomic groups:</div><div class = "text-block">1. Invertebrates</div><div class = "text-block">2. Mammals</div><div class = "text-block">3. Plants</div><div class = "text-block">4. Environmental DNA</div><div class = "text-block">We isolated amplicons using markers for:</div><div class = "text-block"><span>1. rDNA (see Krehenwinkel, H., Pomerantz, A., Henderson, J.B., Kennedy, S.R., Lim, J.Y., Swamy, V., Shoobridge, J.D., Patel, N.H., Gillespie, R.G. and Prost, S., 2018. Nanopore sequencing of long ribosomal DNA amplicons enables portable and simple biodiversity assessments with high phylogenetic resolution across broad taxonomic scale.</span><span style = "font-style:italic;">bioRxiv</span><span>, p.358572.)</span></div><div class = "text-block"><span>2. COI using a mammal cocktail as recommended in Kress, W.J. and Erickson, D.L., 2012. DNA barcodes: methods and protocols. In</span><span style = "font-style:italic;">DNA Barcodes</span><span>(pp. 3-8). Humana Press, Totowa, NJ.</span></div><div class = "text-block">3. rBCL and matK for plant samples</div></div>

[STEPS]
?. [PCRMixes]
1We used a master mix (See below) to set up PCR reactionsButterfly rDNA (Total vol = )each of the forward and reverse primer set l water DNA q5 MasterMixMammal rDNA (Total vol = )each of the forward and reverse primer set l water DNA q5 MasterMixPlant rDNA (Total vol =  )each of the forward and reverse primer set l water DNA q5 MasterMixButterfly COI (Total vol = )each of the forward and reverse primer set l water DNA q5 MasterMixPlant rBCL and matK (Total vol = )each of the forward and reverse primer set l water DNA q5 MasterMixEctoparasites, eDNA, dung beetles, snails COI  (Total vol = ) Taq buffer MGCl2 dNTPs (10mM)each of the forward and reverse primer set l water DNADung beetles, ectoparasites, butterflies (Total vol = ) Taq buffer MGCl2 dNTPs (10mM)each of the forward and reverse primer set l water DNA
25 µl
1 µl
9 µl
[template]
10 µl
13 µl
1 µl
2 µl
[template]
6 µl
13 µl
1 µl
2 µl
[template]
6 µl
25 µl
1 µl
7 µl
[template]
12 µl
25 µl
1 µl
7 µl
[template]
12 µl
12 µl
2 µl
1 µl
0 µl
0 µl
6 µl
[template]
[Hotstart GoTaq]
12 µl
2 µl
2 µl
0 µl
0 µl
5 µl
[template]
[Hotstart GoTaq]
?. [PCR conditions]
rDNA PCR conditionsInitial denaturation for  at Denaturation for  at Annealing for  at Extension for  at Repeat cycles 35 timesFinal extention for   at  COI  (2 sets of conditions, but run one after the other since our program could not handle 2 sets at once.)Initial denaturation for   at  Denaturation for   at  Annealing for   at  Extension for   at  Repeat cycles 5 timesFinal extention for   at  Initial denaturation for   at  Denaturation for   at  Annealing for   at  Extension for   at  Repeat this set for 35 cyclesFinal extention for  at COI  (2 sets of conditions, but run one after the other since our program could not handle 2 sets at once.)Initial denaturation for  at Denaturation for  at Annealing for  at Extension for  at Repeat cycles 5 timesFinal extention for   at  Initial denaturation for   at  Denaturation for   at  Annealing for   at  Extension for   at  Repeat this set for 35 cyclesFinal extention for  at Initial denaturation for  at Denaturation for  at Annealing for  at Extension for  at Final extention for  at
?. [Normalisation and Quantification]
At this point, our field course was running very short on time, so we omitted a quantus fluorometer check of each successful PCR amplicon. We simply added 5 uL of each amplicon directly into a library pool. We would NOT recommend this system, since it resulted in some highlyg concentrated samples overshadowing others. We recommend normalsing to 50 nM and then pooling so each sample is balanced.
?. [Gel electrophoresis]
EquipmentBlueGel system MiniOne systemCreate .8 - 1.0% agarose 1 gel with 13 combs Measure 1 g of agaroseMix agraose with 100 mL of 1xTBEMicrowave the mixture until agarose is completely dissolved (1-3 min)Pour the agarose gel into the tray with the comb in place. Allow the agarose gel to harden (20-30 min)Create .8 - 1.0% agarose 1 gel with 13 combs Measure 1 g of agaroseMix agraose with 100 mL of 1xTBEMicrowave the mixture until agarose is completely dissolved (1-3 min)Pour the agarose gel into the tray with the comb in place. Allow the agarose gel to harden (20-30 min)Insert the agarose gel into electrophoresis equipment and add 1xTBE buffer until the agarose gel is submerged Spot check withof each sampleMix  of loading dye to  of each sample and load the gel. (If  Green Taq buffer with built in loading dye was used, skip this step).Load  of 100bp ladder into the agarose gel. Turn on the electrode and let the DNA run until the band is identifiable
2 µl
1 µl
2 µl
5 µl
?. [Barcoding PCR]
A barcoding PCR was run to attach barcodes from the 96-barcode kit for the MinION to each sampleWe did not use special PCR mastermix at this stage, using instead a mix similar to that of the PCRs aboveWe used  of each barcode primer and  of every postive PCR amplicon in a total volume of 25 uLWe ran the PCR for amplicons ~ 500bp in length at the following conditions:Initil denaturation of hotstart taq at      for  Denaturation at  for  Annealing at  for  Extension at   for  Total number of cycles - 18We used of each barcode primer and of every postive PCR amplicon in a total volume of 25 uLWe ran the PCR for amplicons ~ 500bp in length at the following conditions:Initil denaturation of hotstart taq at     for Denaturation at for Annealing at for Extension at  for Final Extension at  for
1 µl
2 µl
[extend this to 85s for amplicons 1000bp long]
?. [SPRI cleanup]
Run a SPRI cleanup of the library using your choice of bead purification systems/kits in a 1:1 ratio. Resuspend in the same volume.