Jul 21, 2016
eDNA Fish Metabarcoding PCR; 12S & cytB
- Harriet Johnson
- EvoHull
Protocol Citation: Harriet Johnson 2016. eDNA Fish Metabarcoding PCR; 12S & cytB. protocols.io https://dx.doi.org/10.17504/protocols.io.fckbiuw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 20, 2016
Last Modified: March 21, 2018
Protocol Integer ID: 3180
Abstract
One-step metabarcoding PCR. Used to amplify either mitochondrial 12S or cytochrome B regions from environmental DNA sampled from freshwater to identify fish species. PCR product contains the sequencing primers ready for sequencing on the Illumina MiSeq (custom oligos required for sequencing).
Guidelines
References to primer sequences and their use with this method:
Kocher, Thomas D., et al. "Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers." Proceedings of the National Academy of Sciences 86.16 (1989): 6196-6200.
Riaz, Tiayyba, et al. "ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis." Nucleic Acids Research (2011): gkr732.
Kelly, Ryan P., et al. "Using environmental DNA to census marine fishes in a large mesocosm." PloS one 9.1 (2014): e86175.
Hänfling, Bernd, et al. "Environmental DNA metabarcoding of lake fish communities reflects long‐term data from established survey methods." Molecular ecology (2016).
Safety warnings
Custom oligos are required for Illumina sequencing of this PCR product. In order to also sequence PhiX on the run, spike in the custom oligos to the supplied standard Illumina sequencing primers.
The sequencing library preparation protocol will also be added here. I have put a link to the Schloss library prep protocol for now (see links).
Dual indexed primers:
16 forward primers, 24 reverse primers: FA, FB, RA, RB. Can mix and match to create 384 unique combinations to identify multiplexed samples. See 'guidelines' for references to primer sequences.
Dilute primers to 10 uM concentration with water.
Plate primers in matrix on 96 well plate or strip tubes to create unique combinations:
eg.
RA701 ----> RA712
FA501
|
FA508
Note
Mulitple dilutions are recommended for making working concentration primers to minimise handling of stock primers and to avoid contamination.
Expected amplicon size:
Cytochrome B: 460 bp region (F primer: 74, R primer: 70), total: 604 bp
12S: 106 bp region (F primer: 67, R primer: 62), total: 235 bp
Note
Positive control: Rhamphochromis esox, a pelagic species in its natural habitat and isn't common in UK aquariums (according to Alan Smith). Info from Lynsey Harper.
SEPA Jan/Feb 2016: 2 µl 1/100 dilution DNA.
EA, April/May 2016 LFC project: 0.5 ul 1/500 dilution DNA.
Negative control: Molecular grade water
Note
No. of cycles were reduced from 40x to 35x, told of 'updated' protocol when I started, do not know when this was changed.
All rxns I have performed have been 35x.
PCR reaction setup
20 ul rxn: Windermere samples:
10 µl Q5 2x mastermix
1 µl F primer
1 µl R primer
2 µl DNA
6 µl H2O
20 ul rxn: Scottish Loch samples:
10 µl Q5 2x mastermix
1 µl F primer
1 µl R primer
4 µl DNA
4 µl H2O
35ul rxn: LFC samples:
17.5 µl Q5 2x mastermix
1.75 µl F primer
1.75 µl R primer
3.5 µl DNA
10.5 µl H2O
PCR strip tubes with individual lids, instead of open plates.
Note
Mulitple dilutions are recommended for making working concentration primers to minimise handling of stock primers and to avoid contamination.
Expected amplicon size:
Cytochrome B: 460 bp region (F primer: 74, R primer: 70), total: 604 bp
12S: 106 bp region (F primer: 67, R primer: 62), total: 235 bp
Note
Positive control: Rhamphochromis esox, a pelagic species in its natural habitat and isn't common in UK aquariums (according to Alan Smith). Info from Lynsey Harper.
SEPA Jan/Feb 2016: 2 µl 1/100 dilution DNA.
EA, April/May 2016 LFC project: 0.5 ul 1/500 dilution DNA.
Negative control: Molecular grade water
Note
No. of cycles were reduced from 40x to 35x, told of 'updated' protocol when I started, do not know when this was changed.
All rxns I have performed have been 35x.
Thermal cycling parameters:
Initial denaturation: 98°C 5 min;
35X of
1. 98°C 10 sec
2. 58 °C 15 sec
3. 72°C 20 sec;
Final extension 72°C 5 min
Store at 10°C
Note
Mulitple dilutions are recommended for making working concentration primers to minimise handling of stock primers and to avoid contamination.
Expected amplicon size:
Cytochrome B: 460 bp region (F primer: 74, R primer: 70), total: 604 bp
12S: 106 bp region (F primer: 67, R primer: 62), total: 235 bp
Note
Positive control: Rhamphochromis esox, a pelagic species in its natural habitat and isn't common in UK aquariums (according to Alan Smith). Info from Lynsey Harper.
SEPA Jan/Feb 2016: 2 µl 1/100 dilution DNA.
EA, April/May 2016 LFC project: 0.5 ul 1/500 dilution DNA.
Negative control: Molecular grade water
Note
No. of cycles were reduced from 40x to 35x, told of 'updated' protocol when I started, do not know when this was changed.
All rxns I have performed have been 35x.
Visualise PCR product via gel electrophoresis:
large gel:
1.5g agarose
100 ml 0.5x TBE
10 ul GelRed
Run at 90V 40 mins.
2-3 ul product
1.5ul Easy Ladder
Triplicate repeat
SEPA Jan/Feb 2016:
20 ul rxns, performed in triplicate repeat and then pooled.