TITLE: 18S Metagenomics in a Field Setting
AUTHORS: Stefan Prost, Gideon Erkenswick, Mrinalini Watsa, Aaron Pomerantz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is was used to conduct DNA 18S metagenomics on FPI's Genomics in the Jungle - 2018 field course at the Green Lab, located and Inkaterra Guides Field Station, Madre de Dios, Peru.</div></div>

[STEPS]
?. 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
?. [Amplification]
Remove samples and the following reagents and let thaw, once thawed keep on ice block10mM DNTPs25 mMgCl5x Go Taq BufferForward primer 5’-CAGCAGCCGCGGTAATTCC-3’ (10uM)Reverse primer 5’-CCCGTGTTGAGTCAAATTAAGC-3’ (10uM)GoTaq Hotstart Polymerase 5u/ulMake PCR cocktail for # of samples * 1.1 (10% extra). Don't forget to include 1 PCR negative control for each separate PCRRun PCR according to the following cycle conditions:Initial denaturation for 120s25 cycles of for , for , for Final extension  for Upon completion remove, label, and store at , or take directly to electrophoresis
?. [Quantification]
EquipmentdsDNA dye Qubit Assay Tubes Your DNA sample Pulse vortex your sample and spin down.Add   of dsDNA dye into the qubit assay tube.Make sure to cover up the tube from light Transfer  of DNA sample into qubit assay tube Pulse vortex and spin downLet it sit for   in room temperature Covered from the light Pulse vortex your sample and spin down.Add  of dsDNA dye into the qubit assay tube.Make sure to cover up the tube from light Transfer  of DNA sample into qubit assay tube Pulse vortex and spin downLet it sit for   in room temperature Covered from the light Transfer of DNA sample into qubit assay tube Pulse vortex and spin downLet it sit for  in room temperature Gently vortex and spindown the sample Calibrate the Quantus Fluormeter by standard and reference testing.Follow the same process but use  ddH2O and Lambda DNA.Insert the qubit assay tube into the Quantus Flurometer
200 µl
1 µl
1 µl
?. [Normalisation and Pooling]
Dilute each sample to 50 nMMix 5 uL of the sample with calculated amound of ddH2O (based on DNA concentration above) to make each sample the same concentration of 50 nMThen pool 5 uL of each dilution into a single tube.This is now your library.
?. [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.
?. [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 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 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