TITLE: Environmental DNA (eDNA) 12S metabarcoding Illumina MiSeq NGS (2-step) PCR Protocol
AUTHORS: Kathleen Pitz, Kristine Walz

[DESCRIPTION]
This protocol is aimed at amplifying the 12S rRNA gene in eukaryotes. The primers (Mifish_U) utilized in this protocol are based on the primers utilized in Miya et al., 2015. This protocol was used to process data historically but is no longer in use.

12S MiFish_U (forward): 
GTC GGT AAA ACT CGT GCC AGC
 
12S MiFish_U (reverse): 
CAT AGT GGG GTA TCT AAT CCC AGT TTG
 
Amplicons generated using this protocol can then be sequenced using the Illumina platform.
 
Primers used (at MBARI):
Fluidigm CS1+mifish_U (forward)
Fluidigm CS2+mifish_U (reverse)
 
Secondary 12S PCR primers (at MSU):
PE1-BC-CS1
PE2-BC-CS2 

 
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.

[BEFORE_START]
Disinfect work surfaces with 10% bleach, followed by 70% ethanol, then RNase Away, and clean pipets with RNase Away. UV pipets, molecular grade water, and tube racks for 20 minutes prior to starting protocol.

[GUIDELINES]
 Local containment involves the employment of biological safety cabinets for initial specimen disinfection and DNA extraction. Such cabinets should not be used for PCR setup, as this procedure should be conducted apart from the aforementioned disinfection and extraction to minimize contamination.
  
  Do not use any tube or plate that is not appropriate for the PCR machine you are using.
 Make sure tubes and especially plates are well sealed before you begin run.
 Clean up any spilled solutions and dispose of in appropriate biohazard boxes.
 Be careful with PCR machine lids. These can be damaged if you slam or drop lids.
 Make sure PCR heater block is clean before you start a run. Check each tube receptacle before you start.
 Distribute tubes evenly across block so lid will seat flat against top of tubes for even heating and sealing.
 Turn PCR machine off when you are done using.
  

[STEPS]
SECTION: PCR
1. PCR reactions for 12S were run with Fluidigm two-step amplification protocol for each sample. 

SECTION: Primary PCR
2. Primary PCR amplifications were carried out in triplicate 25-μl reactions using: 
1 μl DNA extract
12.5 μl AmpliTaq Gold Fast PCR master mix (Applied Biosystems)
1 μl each of forward and reverse primers (10 μM) 
9.5 μl molecular-biology grade water 
SECTION: Primary PCR
3. PCR reactions were run in 96-well plates with a NTC run in triplicate for each plate. 

SECTION: Primary PCR
4. Primary 12S PCR cycling parameters:
95 °C for 10 minutes
40 cycles of the following three steps:
95 °C for 15 seconds
55 °C for 30 seconds
72 °C for 30 seconds
A final elongation step of 72 °C for 10 minutes
Hold at 4 °C
 
SECTION: Primary PCR clean-up
6. After primary PCR amplification of the marker region, the pooled PCR products were run through an agarose gel to confirm the presence of target bands and absense of non-specific amplification across environmental samples as well as the absence of amplification in no-template controls (NTCs).
SECTION: Secondary PCR
10. Secondary PCR amplifications were carried out as single 15-μl reactions using:
1 μl template of primary PCR product (1:75 dilution) 
6 μl OneTaq Hot Start 2X master mix with standard buffer (NEB)
1 μl of forward and reverse primer mix (6 μM) 
7 μl molecular-biology grade water 
SECTION: Secondary PCR
11. Secondary COI cycling parameters:
95 °C for 3 minutes 
15 cycles of the following three steps:
95 °C for 15 seconds 
60 °C for 30 seconds 
72 °C for 60 seconds 
Then a final elongation step of 72 °C for 3 minutes 
Hold at 25 °C
SECTION: Secondary PCR
12. Secondary Fluidigm PCR primers (primers listed in 5’ to 3’ direction)
PE1-BC-CS1 (forward):
AATGATACGGCGACCACCGAGATCT-[i5-BC(index 2)]-ACACTGACGACATGGTTCTACA
PE2-BC-CS2 (reverse):
CAAGCAGAAGACGGCATACGAGAT-[i7-BC(index 1)]-TACGGTAGCAGAGACTTGGTCT
SECTION: Quality control, PCR clean-up and sequencing parameters
13. An agarose gel was run after secondary PCR to confirm the presence of target bands and absense of non-specific amplification across environmental samples as well as the absence of amplification in no-template controls (NTCs).
SECTION: Quality control, PCR clean-up and sequencing parameters
14. After secondary PCR, products were run through Invitrogen SequalPrep Normalization Plate (ThermoFisher Scientific) using manufacturer's protocol to create pooled library.
SECTION: Sequencing
15. 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.
SECTION: Sequencing
16. The MiSeq run was performed with a 10% PhiX spike added.
SECTION: Sequencing
17. 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.
SECTION: Sequencing
18. 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 
SECTION: Sequencing
19. 12S Sequencing primers (5’ to 3’ direction):
FL1-CS1(read1) A+CA+CTG+ACGACATGGTTCTACA
FL1-CS2(read2) T+AC+GGT+AGCAGAGACTTGGTCT
FL2-CS1rc          T+GT+AG+AACCATGTCGTCAGTGT
FL2-CS2rc(index)   A+GAC+CA+AGTCTCTGCTACCGTA
SECTION: Sequencing
20.       Sequencing is performed at the Research Technology Support Facility (RTSF) Genomics Core at Michigan State University (MSU).
 
 
 
 
 
 
SECTION: Primary PCR clean-up
7. Primary PCR products were purified and size selected using the Agencourt AMPure XP bead system (Beckman Coulter, USA). 
SECTION: Secondary PCR
9. 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. 
SECTION: Primary PCR
5. 12S Primary PCR primers (primers listed in 5’ to 3’ direction)
Fluidigm CS1+MiFish_U(forward):
ACACTGACGACATGGTTCTACA GTCGGTAAAACTCGTGCCAGC
Fluidigm CS2+Mifish_U(reverse):
TACGGTAGCAGAGACTTGGTCT CATAGTGGGGTATCTAATCCCAGTTTG
SECTION: Primary PCR clean-up
8. A second agarose gel was run to confirm primer removal and retention of target amplicons after purification.