TITLE: Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS PCR Protocol (Touchdown)
AUTHORS: Katie Pitz, Nathan Truelove, Charles Nye, Reiko P Michisaki, Francisco Chavez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The 12S protocol is aimed at amplifying the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. The primers (MiFish-U-F & MiFish-U-R) used in this protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes. </div><div class = "text-block">Touchdown thermocycling protocols were adapted from the CALeDNA group.</div><div class = "text-block">This work was supported by NASA grant NNX14AP62A ‘National Marine Sanctuaries as Sentinel Sites for a Demonstration Marine Biodiversity Observation Network (MBON)’ funded under the National Ocean Partnership Program (NOPP RFP NOAA-NOS-IOOS-2014-2003803 in partnership between NOAA, BOEM, and NASA), and the U.S. Integrated Ocean Observing System (IOOS) Program Office.</div><div class = "text-block">Citations</div><div class = "text-block">1) Miya M et al. 2015 MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. R.Soc.opensci. 2: 150088. http://dx.doi.org/10.1098/rsos.150088</div><div class = "text-block">2) CALeDNA. 2019. University of California Conservation Genomics Consortium.</div></div>

[STEPS]
?. [Setup and Primary PCR]
eDNA template & PCR processing were performed at the Monterey Bay Aquarium Research Institute (MBARI).PCR reactions for the 12S locus were performed with a two-step amplification protocol for each sample using the MiFish_U primers (Miya et al. 2015) with Fluidigm adapters CS1 & CS2.All primers listed in the 5’ to 3’ direction. MiFish primers are in bold.Fluidigm CS1 + 12S MiFish_U (forward): ACACTGACGACATGGTTCTACAGTCGGTAAAACTCGTGCCAGCFluidigm CS2 + 12S MiFish_U (reverse): TACGGTAGCAGAGACTTGGTCTCATAGTGGGGTATCTAATCCCAGTTTG
?. [Setup and Primary PCR]
The primary PCR amplifications were carried out in triplicate 25 μl reactions using:1 μL eDNA extract template 12.5 μL AmpliTaq Gold™ Fast PCR Master Mix1 μL forward primer (10 μM)1 μL reverse primer (10 μM)9.5 μL molecular-grade, nuclease-free water
?. [Setup and Primary PCR]
PCR reactions were performed in 96-well plates with a no-template control (NTC) for each PCR plate, for a total of 3 PCR negative controls. An artificial community was used as a positive control.
?. [Setup and Primary PCR]
Primary 12S cycling parameters, using the CALeDNA Touchdown method:1. 95°C 15 minutes*13 cycles of the following 3 steps (step 3 changes -1.5°C each cycle; "touchdown")2. 94°C 30 seconds*3. 69.5°C 30 seconds*4. 72°C 90 seconds**25 cycles of the following 3 steps5. 94°C 30 seconds*6. 50°C 30 seconds*7. 72°C 45 seconds*8. 72°C 10 minutes9. 4°C HOLD
?. [Quality Control and Product Cleanup]
After primary PCR amplification of the marker region, the PCR products were pooled (75 μL total per unique environmental sample) and run through a 2% agarose gel to confirm the presence of target bands and absense of non-specific amplification across environmental samples.
?. [Quality Control and Product Cleanup]
Primary PCR products were purified and size selected using the Agencourt AMPure XP bead system (Beckman Coulter, USA) at 1.2x volume beads to product.
?. [Quality Control and Product Cleanup]
A second agarose gel was run to confirm primer removal and retention of target amplicons after purification. NTCs were also tested using a Qubit dsDNA 1x high sensitivity kit to ensure no amplification.
?. [Secondary Amplification]
Secondary amplification and NGS were performed at Michigan State University's Research Technology Support Facility (RTSF).An aliquot of 20 μL from each purified primary PCR product was sent to RTSF Genomics Core at MSU for secondary PCR amplification with primers which targeted the CS1/CS2 ends of the primary PCR products and added dual indexed, Illumina compatible adapters with barcodes. PE1-BC-CS1 (forward):AATGATACGGCGACCACCGAGATCT-[i5-BC(index 2)]-ACACTGACGACATGGTTCTACAPE2-BC-CS2 (reverse):CAAGCAGAAGACGGCATACGAGAT-[i7-BC(index 1)]-TACGGTAGCAGAGACTTGGTCT
?. [Secondary Amplification]
The secondary PCR amplifications were carried out in 15 μL reactions, using 1 μL original pooled PCR product triplicates.6 μl 2.5X HotMaster Mix7 μl DI water1 μl Primer Mix (6uM)1 μl original eDNA PCR product
?. [Secondary Amplification]
Secondary 12S cycling parameters:1. 95°C 3 minutes*15 cycles of the following 3 steps2. *95°C 15 seconds3. *60°C 30 seconds4. *72°C 1 minute5. 72°C 3 minutes6. 25°C Hold
?. [Secondary Amplification]
An agarose gel was run after secondary PCR to confirm the presence of target bands and absence of non-specific amplification across environmental samples as well as the absence of amplification in NTCs.
?. [Secondary Amplification]
After secondary PCR, products were run through Invitrogen SequalPrep Normalization Plate (ThermoFisher Scientific) using manufacturer's protocol to create pooled library.
?. [Sequencing]
The pooled product for the genetic locus was loaded on a standard MiSeq v2 flow cell and sequenced in a 2x250bp paired end format using a v2 500-cycle MiSeq reagent cartridge.
?. [Sequencing]
The MiSeq run was performed with a 10% PhiX spike added.
?. [Sequencing]
Primers complementary to the Fluidigm CS1 & CS2 oligomers were added to appropriate wells of the reagent cartridge to server as sequencing and index read primers.
?. [Sequencing]
Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.18.0
?. [Sequencing]
12S Sequencing primers (5’ to 3’ direction):FL1-CS1(read1)	A+CA+CTG+ACGACATGGTTCTACAFL1-CS2(read2)	T+AC+GGT+AGCAGAGACTTGGTCTFL2-CS1rc		T+GT+AG+AACCATGTCGTCAGTGTFL2-CS2rc(index)	A+GAC+CA+AGTCTCTGCTACCGTA