Jul 15, 2022
Non-destructively barcoding hundreds of freshwater macroinvertebrates with a MinION
- 1Randolph-Macon College
Protocol Citation: Elise C. Knobloch, Ray C Schmidt 2022. Non-destructively barcoding hundreds of freshwater macroinvertebrates with a MinION. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzy974lx1/v1
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
We use this protocol and it’s working
Created: June 17, 2022
Last Modified: July 15, 2022
Protocol Integer ID: 64798
Keywords: Barcoding, Freshwater macroinvertebrates, non-destructive, biodiversity, CO1, Nanopore, Flongle
Funders Acknowledgement:
National Science Foundation
Grant ID: 1920116
Abstract
This project aimed to optimize protocols needed to produce CO1 barcodes for 1000s of African freshwater macroinvertebrates, from many different orders, in the most cost-efficient way possible. Since many of these specimens represent undescribed or poorly-known taxa we also wanted to utilize a non-destructive method of DNA extraction. To do so, we modified the methods detailed by Srivathsan et al. (2021). Here we present the protocol from specimen preparation and DNA extraction to sequence generation. In addition to the methods outlined by Srivathsan et al. (2021) we also pulled together the protocols from Oxford Nanopore Technologies and other vendors. We have added some tips and comments to these procedures that we found helpful in the process. We used these protocols to produce barcodes for hundreds of freshwater macroinvertebrates that were collected in Gabon. This project was funded by the National Science Foundation's Research Experience for Post-Baccalaureate Students (REPS) program (DEB #1920116).
CITATION
Image Attribution
Ray C. Schmidt
Guidelines
These protocols are modified from those described in Srivathsan et al 2021.
CITATION
Materials
Equipment
MinION
NAME
Sequencer
TYPE
Oxford Nanopore Technologies
BRAND
MinION 1B / MinION 1C
SKU
Equipment
Flongle adaptor
NAME
Oxford Nanopore Technologies
BRAND
ADP-FLG001
SKU
QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647 NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646 AMPure XP Beckman CoulterCatalog #A63880 Adapter Mix (AMX)Oxford Nanopore Technologies NEBNext Quick T4 DNA LigaseNew England Biolabs Ligation Buffer (LNB)Oxford Nanopore Technologies Elution Buffer (EB)Oxford Nanopore Technologies Short Fragment Buffer (SFB)Oxford Nanopore Technologies FB (Flush Buffer)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 FLT (Flush Tether)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 Sequencing Buffer II (SBII)Oxford Nanopore Technologies Loading Beads II (LBII)Oxford Nanopore Technologies 2.0X Taq RED Master MixGenesee ScientificCatalog #42-138 QuantiFluor ONE dsDNA DyePromega
Equipment
T100 thermocycler
NAME
BioRad
BRAND
1861096
SKU
Equipment
blueGel
NAME
MiniPCR
BRAND
QP-1500-01
SKU
Equipment
Quantas Fluorometer
NAME
Fluorometer
TYPE
Promega
BRAND
E6150
SKU
Protocol materials
QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
In Materials and 2 steps
Adapter Mix (AMX)Oxford Nanopore Technologies
Materials, Step 5.1
NEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
Materials, Step 4.2
FB (Flush Buffer)Oxford Nanopore TechnologiesCatalog #EXP-FLP002
Materials, Step 7.1
2.0X Taq RED Master MixGenesee ScientificCatalog #42-138
Materials, Step 2.1
Elution Buffer (EB)Oxford Nanopore Technologies
In Materials and 2 steps
NEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
Materials, Step 4.2
Short Fragment Buffer (SFB)Oxford Nanopore Technologies
In Materials and 3 steps
Ligation Buffer (LNB)Oxford Nanopore Technologies
Materials, Step 5.1
NEBNext Quick T4 DNA LigaseNew England Biolabs
Step 5.2
AMPure XP Beckman CoulterCatalog #A63880
In Materials and 5 steps
FLT (Flush Tether)Oxford Nanopore TechnologiesCatalog #EXP-FLP002
Materials, Step 7.1
QuantiFluor ONE dsDNA DyePromega
Materials, Step 3.18
Sequencing Buffer II (SBII)Oxford Nanopore Technologies
Materials, Step 7.2
NEBNext Quick T4 DNA LigaseNew England Biolabs
Materials, Step 5.1
Loading Beads II (LBII)Oxford Nanopore Technologies
Materials, Step 7.2
Specimen preparation and DNA extraction
Specimen preparation and DNA extraction
20m
20m
Specimen preparation and DNA extraction
Specimen preparation
Specimen preparation
20m
20m
Fill two Petri dishes with ddH2O
Remove specimen/s from ethanol and place in one dish of ddH2O for00:10:00
Note
You may need to work under a microscope depending on what type of specimens you are working with.
Note
If you are removing specimens from a jar, be sure to top off that jar with ethanol when you are finished.
10m
Remove specimen/s from water, and rinse again in the second dish for 00:05:00
5m
Place specimen/s on a paper towel to dry for about 00:05:00
Note
Arranging the specimens in rows of eight makes the next steps easier.
5m
DNA extraction
DNA extraction
20m
20m
Put 10 µL QuickExtract DNA Extraction SolutionBio-rad LaboratoriesCatalog #QE09050 in each well of a 96-well plate
Note
I have found that working in rows of three is the most effective and reduces the likelihood to make mistakes
Place one rinsed and dried specimen in each well head-first
Note
Be sure the head is fully submerged. If the specimen is too large to fit in the well, remove a leg and place that in the quick extract.
Note
When working with legs, be sure to keep one side of the specimen intact
Note
This was the work flow I found to be most effective: remove 24 specimens at a time and place them in the water to soak. Fill three rows of the plate with QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 . Remove specimens individually from the water and place in 3 rows of 8 to dry out (they do not need to dry for long). Place each specimen in a well. Put clean water in both dishes (trying to prevent ethanol from being a PCR inhibitor). Repeat this process until the plate is full.
Cover with TempPlate sealing foil or reusable TempPlate pressure-fit sealing mat
Place the covered 96-well plate in the thermal cycler
Note
Seal plate as well as you can when heating, QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 will start to evaporate.
Heat at 65 °C for 00:18:00 ,98 °C for 00:02:00
20m
After heating, remove theQuickExtract DNA Extraction SolutionBio-rad LaboratoriesCatalog #QE09050 from each well and transfer to a clean 96-well plate
Note
- Be very careful to not touch specimens when removing QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 from the wells containing the specimens. Some soft-bodied specimens may disintegrate and others may be very fragile and should not be touched
- Some hard-bodied specimens tend to soak up the QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 . If you go to remove the QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 from that well and there isn’t any liquid in the well, add 10 µL of ddH2O, pipette up and down, and remove this as your extract.
- Move slowly when doing this, it is easy to get mixed up on which well you are on when dealing with a full 96 well plate
DNA Dilution
DNA Dilution
Put 10 µL of ddH2O in each well of a 96-well plate
Put 1 µL of DNA extraction in each coinciding well
Note
- We have found that the concentration of the quick extract DNA extract can range anywhere between about 1ng/mL to about 50ng/mL. Diluting the DNA 1:10 has been the most successful generalized protocol (about 75% PCR success)
- Seal the dilution plates well when storing, they will evaporate if not
PCR Amplification
PCR Amplification
PCR Amplification
PCR Setup
PCR Setup
Combine ddH2O, 2.0X Taq RED Master MixBio-rad LaboratoriesCatalog #42-138 , and forward primer in a tube
| A | B | |
| Per 20µL Reaction | ||
| ddH20 | 7 | |
| 2x Buffer-APEX red | 10 | |
| Primer F | 0.5 | |
| Primer R | 0.5-not in master mix | |
| DNA Template | 2-not in master mix |
Note
We have 12 forward primers and 24 reverse primers (purchased from Integrated DNA Technologies, Inc.) so I typically work with 24 wells at a time. I make the master mix for 25x to make sure I have enough master mix to distribute into each tube.
| 25x | ||
| H20 | 175 | |
| 2x Buffer-APEX red | 250 | |
| Primer F | 12.5 | |
| Primer R | 0.5-not in mastermix | |
| DNA Template | 2-not in mastermix |
Primer List.xlsx Distribute 17.5 µL of master mix into each tube of a strip tube
Note
Work on an ice block because this setup takes anywhere from 00:10:00 to 00:20:00
Distribute DNA template and reverse primer into each tube
Note
Workflow: in a 1.5 ml tube, combine the 25x amount of the water, 2.0X Taq RED Master MixGenesee ScientificCatalog #42-138 , and forward primer that you are using. Vortex and spin down. In three 8-tube strip tubes, distribute 17.5 µL of the mastermix. Each separate tube gets 0.5 µL of a unique reverse primer. Each separate tube gets 2 µL of a unique specimen’s diluted DNA. Add the DNA and reverse primers in order to be able to keep track more easily.
Thermal Cycler Protocol:94 °C for 00:01:00 , then 94 °C for 00:00:30 , 42 °C for 00:01:00 , 72 °C for 00:01:30 , go back to step 2 39x, 72 °C for 00:07:00 , infinite hold at 10 °C
Equipment
T100 Thermal Cycler
NAME
Thermal Cycler
TYPE
BioRad
BRAND
1861096
SKU
11m
Gel Electrophoresis
Gel Electrophoresis
Run a gel on at least 6 specimens from each master mix batch. Including a subset of the reaction on the gel allows you to confirm that there were no issues with the PCR master mix and also estimates how many reactions were successful.
Expected result
Running a subset of reaction on a gel to confirm successful amplification, allows estimation of success rate (e.g. ~66%)
Pooling and Cleaning
Pooling and Cleaning
Pooling and cleaning
Take 4 µL from each PCR-This will yield approximately 1.152 mL of PCR product
Take 250 µL from the pooled PCR
Expected result
Unpurified pooled PCR reactions
Add 125 µL of AMPure XP Bio-rad LaboratoriesCatalog #A63880 and pipette up and down to mix (Followed manufacturer protocol but listed here).
Note
We used 0.5 X AMPure beads
Let sit for 00:10:00 at room temperature
10m
Let sit on magnetic rack for 00:02:00
Note
Keep the tube open, jolting the tube when closing and opening the lid can disturb the pellet
2m
Remove supernatant, leaving about 5 µL on the bottom of the tube
Add 250 µL of freshly made 70% ethanol and pipette gently up and down
Let sit on rack for 00:00:30
30s
Remove all ethanol
Add another 250 µL of 70% ethanol and let sit for 00:00:30
30s
Remove all ethanol
Let dry for 00:05:00 to00:10:00 with the cap open so the ethanol can evaporate
15m
Add 40 µL of ddH2O and elute the pellet (remove from the rack)
Let sit for 00:02:00
2m
Put back on the rack and let pellet form
Expected result
pelleted PCR product
Remove liquid away from pellet-->this is our cleaned product
Expected result
purified PCR product on right
Run in a gel compared to uncleaned product to evaluate success
Expected result
First column is uncleaned product, second column is cleaned product, third column is 100bp ladder
·The clean was successful if the cleaned product does not have any bands below the Co1 band
Quantify cleaned PCR with
Equipment
Quantas Fluorometer
NAME
Fluorometer
TYPE
Promega
BRAND
E6150
SKU
Qubit Fluorometer (QuantasTM Fluorometer protocol listed below)
1. Mix 1 µL of DNA sample with 200 µL of QuantiFluor ONE dsDNA DyeBio-rad Laboratories in a 0.5ml PCR tube.
2. Vortex
3. Place tube into the tube holder and close the lid
4. Vortex sample again and repeat to be sure you got an accurate reading
Library preparation
Library preparation
10m
10m
Library preparation
Note
Make sure to check flow cells before starting this step. There is a two-week lead time for Flongle flow cells so plan accordingly.
Dilute DNA-->concentration of DNA should be 100-200fmol amplicon DNA
Note
The protocol listed below is provided by Oxford Nanopore Technologies, modified based on the recommendations given in Srivathsan et al. 2021.
CITATION
amplicon-sqk-lsk109_protocol.pdf Note
·When the concentration of our pooled and cleaned product was 41 ng/µL , we did 21 µL of water and 1.5 µL of DNA.
·When the concentration of the pooled and cleaned product was about 80 ng/µL and about 94 ng/µL , we did 21.5 µL of water, 1 µL of DNA
Combine in a strip-tube: 22.5 µL of diluted DNA, 3.5 µL of NEBNext Ultra II End Prep Reaction BufferBio-rad LaboratoriesCatalog #E7647 , 1.5 µL of NEBNext Ultra II End Prep Enzyme MixBio-rad LaboratoriesCatalog #E7646 , and 2.5 µL of H2O
Heat at 20 °C for 00:05:00 , 65 °C for 00:05:00
10m
Transfer mix into new 1.5 ml tube
Vortex AMPure XP Bio-rad LaboratoriesCatalog #A63880
Add 30 µL of AMPure XP Bio-rad LaboratoriesCatalog #A63880 to the mixture
Incubate on Hula mixer for 00:05:00
5m
Spin down, place on magnet until clear
Keeping the tube on the magnet, pipette off supernatant
Add 100 µL of 70% ethanol (do not touch pellet)
Remove ethanol
Add 100 µL of 70% ethanol, take off magnet rack and spin down, put back on magnetic rack
Remove ethanol, allow to dry for about 00:00:30
30s
Add 30.5 µL of ddH2), remove from magnetic rack and resuspend pellet
Incubate off rack for 00:02:00
2m
Place back on magnetic rack until clear
Remove liquid and put in clean tube
Library preparation: Ligation
Library preparation: Ligation
10m
10m
Ligation
Spin downAdapter Mix (AMX)Bio-rad Laboratories and NEBNext Quick T4 DNA LigaseBio-rad Laboratories and put on ice. Thaw and vortex Ligation Buffer (LNB)Bio-rad Laboratories and put on ice, thaw and mix Elution Buffer (EB)Bio-rad Laboratories (EB)and put on ice, thaw Short Fragment Buffer (SFB)Bio-rad Laboratories (SFB), vortex, put on ice
Combine in a new tube in this order: 30 µL of DNA from previous step, 12.5 µL of LNB, 5 µL of NEBNext Quick T4 DNA LigaseBio-rad Laboratories , and 2.5 µL of AMX
Flick and spin mixture, incubate at room temp for 00:10:00
10m
Vortex AMPure XP Bio-rad LaboratoriesCatalog #A63880
Add 20 µL of AMPure XP Bio-rad LaboratoriesCatalog #A63880 to mixture and flick tube
Incubate at room temp on Hula mixer for 00:05:00
5m
Spin sample, put on magnetic rack, let pellet form, pipette of supernatant
Add 125 µL of Short Fragment Buffer (SFB)Bio-rad Laboratories , flick tube to resuspend, spin down, return tube to magnetic rack, let pellet form, remove supernatant
Add 125 µL of Short Fragment Buffer (SFB)Bio-rad Laboratories , flick tube to resuspend, spin down, return tube to magnetic rack, let pellet form, remove supernatant
Note
·I have found that the pellet does not stick to the magnet as strongly during this step as it does in previous steps, making it harder to pipette off the supernatant. Pushing the tube as close to the magnet as you can and tilting the rack while pipetting the supernatant off helps.
Allow to dry for about 00:00:30
30s
Add 15 µL ofElution Buffer (EB)Bio-rad Laboratories , spin down and resuspend, incubate at room temp for 00:10:00
10m
Place on magnetic rack and allow pellet to form, remove liquid and transfer to a clean tube
Note
·I repeat this step twice to be sure it is fully cleaned
Quantify
Note
Following same quantifying protocol as previously listed
Dilute so the concentration of the DNA library is 3-20fmol
Note
·When the concentration of the DNA library was 1.2 ng/µL , we diluted 3 µL DNA: 2 µL Elution Buffer (EB)Oxford Nanopore Technologies
·When the concentration of the DNA library was 1.4 ng/µL , we diluted 2.9 µL DNA: 2.2 µL Elution Buffer (EB)Oxford Nanopore Technologies
Setting Nanopore Parameters
Setting Nanopore Parameters
10m
10m
Setting Nanopore Sequencing Parameters
We largely followed the default settings for the sequencing run. W set the sequencing kit to SQK-LSK-109, run time of 17 hours, and changed the FastQ so that it didn't compress the files.
Loading flow cell
Loading flow cell
10m
10m
Loading flow cell
Note
Priming buffer: mix 117 µL of FB (Flush Buffer)Bio-rad LaboratoriesCatalog #EXP-FLP002 and 3 µL of FLT (Flush Tether)Bio-rad LaboratoriesCatalog #EXP-FLP002 in a tube
Mix 15 µL of 10 µL Sequencing Buffer II (SBII)Bio-rad Laboratories of Loading Beads II (LBII)Bio-rad Laboratories and 5 µL of DNA library in a separate tube
.Insert 120 µL of the priming buffer into the flow cell
Insert 30 µL of prepped DNA library into flow cell
Demultiplexing
Demultiplexing
10m
10m
Demultiplexing
Note
Full description of these procedures and the different options can be found here: https://github.com/asrivathsan/ONTbarcoder
Creating Demultiplexing file
Creating Demultiplexing file
10m
10m
- Create an excel sheet with 5 columns
- First column: sample/specimen ID
- Second column: forward tag
- Third column: reverse tag
- Fourth column: forward primer sequence
Note
- When filling in sheet, be careful to not insert extra spaces or punctuation
- Columns 4 and 5 should be the same throughout the whole sheet
- If you extracted and amplified 288 specimens, you should have 288 rows
Save as a .txt file
Example demultiplexing text file.txt Merge resulting Fastq files
Note
View resulting CO1 sequences for further analyses.
Citations
Srivathsan A, Lee L, Katoh K, Hartop E, Kutty SN, Wong J, Yeo D, Meier R. ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone.
https://doi.org/10.1186/s12915-021-01141-xSrivathsan A, Lee L, Katoh K, Hartop E, Kutty SN, Wong J, Yeo D, Meier R. ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone.
https://doi.org/10.1186/s12915-021-01141-x