Jul 15, 2022

Public workspaceNon-destructively barcoding hundreds of freshwater macroinvertebrates with a MinION

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzy974lx1/v1
Non-destructively barcoding hundreds of freshwater macroinvertebrates with a MinION
  • Elise C. Knobloch1,
  • Ray Schmidt1
  • 1Randolph-Macon College
Ray C Schmidt
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzy974lx1/v1
Protocol Citation: Elise C. Knobloch, Ray 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, african freshwater macroinvertebrate, hundreds of freshwater macroinvertebrate, freshwater macroinvertebrate, dna extraction to sequence generation, dna extraction, protocols from oxford nanopore technology, protocol from specimen preparation, oxford nanopore, oxford nanopore technology, specimen, many of these specimen, specimen preparation, nanopore, dna, barcodes for hundred, barcode, barcoding hundred, gabon
Funders Acknowledgements:
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
Srivathsan A, Lee L, Katoh K, Hartop E, Kutty SN, Wong J, Yeo D, Meier R (2021). ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC biology.
https://doi.org/10.1186/s12915-021-01141-x
LINK

Image Attribution
Ray C. Schmidt
Guidelines
These protocols are modified from those described in Srivathsan et al 2021.
Citation
Srivathsan A, Lee L, Katoh K, Hartop E, Kutty SN, Wong J, Yeo D, Meier R (2021). ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC biology.
https://doi.org/10.1186/s12915-021-01141-x
LINK

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
ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647 ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646 ReagentAMPure XP Beckman CoulterCatalog #A63880 ReagentAdapter Mix (AMX)Oxford Nanopore Technologies ReagentNEBNext Quick T4 DNA LigaseNew England Biolabs ReagentLigation Buffer (LNB)Oxford Nanopore Technologies ReagentElution Buffer (EB)Oxford Nanopore Technologies ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies ReagentFB (Flush Buffer)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 ReagentFLT (Flush Tether)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 ReagentSequencing Buffer II (SBII)Oxford Nanopore Technologies ReagentLoading Beads II (LBII)Oxford Nanopore Technologies Reagent2.0X Taq RED Master MixGenesee ScientificCatalog #42-138 ReagentQuantiFluor 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
ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
ReagentAMPure XP Beckman CoulterCatalog #A63880
ReagentFB (Flush Buffer)Oxford Nanopore TechnologiesCatalog #EXP-FLP002
Reagent2.0X Taq RED Master MixGenesee ScientificCatalog #42-138
ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
ReagentElution Buffer (EB)Oxford Nanopore Technologies
ReagentLoading Beads II (LBII)Oxford Nanopore Technologies
ReagentQuantiFluor ONE dsDNA DyePromega
ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
ReagentAdapter Mix (AMX)Oxford Nanopore Technologies
ReagentFLT (Flush Tether)Oxford Nanopore TechnologiesCatalog #EXP-FLP002
ReagentSequencing Buffer II (SBII)Oxford Nanopore Technologies
ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies
ReagentNEBNext Quick T4 DNA LigaseNew England Biolabs
ReagentLigation Buffer (LNB)Oxford Nanopore Technologies
ReagentNEBNext Quick T4 DNA LigaseNew England Biolabs
Troubleshooting
Specimen preparation and DNA extraction
20m

Specimen preparation and DNA extraction

Specimen preparation
20m
Fill two Petri dishes with ddH2O
Remove specimen/s from ethanol and place in one dish of ddH2O forDuration00:10:00

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 Duration00:05:00

5m
Place specimen/s on a paper towel to dry for about Duration00:05:00
Note
Arranging the specimens in rows of eight makes the next steps easier.



5m
DNA extraction
20m
Put Amount10 µL ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #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 ReagentQuickExtract 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, ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 will start to evaporate.


Heat at Temperature65 °C for Duration00:18:00 ,Temperature98 °C for Duration00:02:00

20m
After heating, remove theReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 from each well and transfer to a clean 96-well plate
Note
  • Be very careful to not touch specimens when removing ReagentQuickExtract 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 ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 . If you go to remove the ReagentQuickExtract DNA Extraction SolutionLucigenCatalog #QE09050 from that well and there isn’t any liquid in the well, add Amount10 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
Put Amount10 µL of ddH2O in each well of a 96-well plate

Put Amount1 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 Setup

Combine ddH2O, Reagent2.0X Taq RED Master MixGenesee ScientificCatalog #42-138 , and forward primer in a tube
AB
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

Download Primer List.xlsxPrimer List.xlsx

Distribute Amount17.5 µL of master mix into each tube of a strip tube
Note
Work on an ice block because this setup takes anywhere from Duration00:10:00 to Duration00:20:00



Distribute DNA template and reverse primer into each tube
Note
Workflow: in a 1.5 ml tube, combine the 25x amount of the water, Reagent2.0X 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 Amount17.5 µL of the mastermix. Each separate tube gets Amount0.5 µL of a unique reverse primer. Each separate tube gets Amount2 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:Temperature94 °C for Duration00:01:00 , then Temperature94 °C for Duration00:00:30 , Temperature42 °C for Duration00:01:00 , Temperature72 °C for Duration00:01:30 , go back to step 2 39x, Temperature72 °C for Duration00:07:00 , infinite hold at Temperature10 °C

Equipment
T100 Thermal Cycler
NAME
Thermal Cycler
TYPE
BioRad
BRAND
1861096
SKU

11m
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

Take Amount4 µL from each PCR-This will yield approximately Amount1.152 mL of PCR product

Take Amount250 µL from the pooled PCR

Expected result
Unpurified pooled PCR reactions


Add Amount125 µL of ReagentAMPure XP Beckman CoulterCatalog #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 Duration00:10:00 at room temperature

10m
Let sit on magnetic rack for Duration00:02:00
Note
Keep the tube open, jolting the tube when closing and opening the lid can disturb the pellet


2m
Remove supernatant, leaving about Amount5 µL on the bottom of the tube

Add Amount250 µL of freshly made 70% ethanol and pipette gently up and down

Let sit on rack for Duration00:00:30

30s
Remove all ethanol
Add another Amount250 µL of 70% ethanol and let sit for Duration00:00:30

30s
Remove all ethanol
Let dry for Duration00:05:00 toDuration00:10:00 with the cap open so the ethanol can evaporate



15m
Add Amount40 µL of ddH2O and elute the pellet (remove from the rack)

Let sit for Duration00:02:00

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 Amount1 µL of DNA sample with Amount200 µL of ReagentQuantiFluor ONE dsDNA DyePromega 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
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
Srivathsan A, Lee L, Katoh K, Hartop E, Kutty SN, Wong J, Yeo D, Meier R (Invalid date). ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC biology.
https://doi.org/10.1186/s12915-021-01141-x
LINK
Download amplicon-sqk-lsk109_protocol.pdfamplicon-sqk-lsk109_protocol.pdf


Note
·When the concentration of our pooled and cleaned product was Concentration41 ng/µL , we did Amount21 µL of water and Amount1.5 µL of DNA.
·When the concentration of the pooled and cleaned product was about Concentration80 ng/µL and about Concentration94 ng/µL , we did Amount21.5 µL of water, Amount1 µL of DNA




Combine in a strip-tube: Amount22.5 µL of diluted DNA, Amount3.5 µL of ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647 , Amount1.5 µL of ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646 , and Amount2.5 µL of H2O

Heat at Temperature20 °C for Duration00:05:00 , Temperature65 °C for Duration00:05:00

10m
Transfer mix into new 1.5 ml tube
Vortex ReagentAMPure XP Beckman CoulterCatalog #A63880

Add Amount30 µL of ReagentAMPure XP Beckman CoulterCatalog #A63880 to the mixture

Incubate on Hula mixer for Duration00:05:00

5m
Spin down, place on magnet until clear
Keeping the tube on the magnet, pipette off supernatant
Add Amount100 µL of 70% ethanol (do not touch pellet)

Remove ethanol
Add Amount100 µL of 70% ethanol, take off magnet rack and spin down, put back on magnetic rack

Remove ethanol, allow to dry for about Duration00:00:30

30s
Add Amount30.5 µL of ddH2), remove from magnetic rack and resuspend pellet

Incubate off rack for Duration00:02:00

2m
Place back on magnetic rack until clear
Remove liquid and put in clean tube
Library preparation: Ligation
10m
Ligation
Spin downReagentAdapter Mix (AMX)Oxford Nanopore Technologies and ReagentNEBNext Quick T4 DNA LigaseNew England Biolabs and put on ice. Thaw and vortex ReagentLigation Buffer (LNB)Oxford Nanopore Technologies and put on ice, thaw and mix ReagentElution Buffer (EB)Oxford Nanopore Technologies (EB)and put on ice, thaw ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies (SFB), vortex, put on ice











Combine in a new tube in this order: Amount30 µL of DNA from previous step, Amount12.5 µL of LNB, Amount5 µL of ReagentNEBNext Quick T4 DNA LigaseNew England Biolabs , and Amount2.5 µL of AMX

Flick and spin mixture, incubate at room temp for Duration00:10:00

10m
Vortex ReagentAMPure XP Beckman CoulterCatalog #A63880

Add Amount20 µL of ReagentAMPure XP Beckman CoulterCatalog #A63880 to mixture and flick tube

Incubate at room temp on Hula mixer for Duration00:05:00

5m
Spin sample, put on magnetic rack, let pellet form, pipette of supernatant
Add Amount125 µL of ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies , flick tube to resuspend, spin down, return tube to magnetic rack, let pellet form, remove supernatant

Add Amount125 µL of ReagentShort Fragment Buffer (SFB)Oxford Nanopore Technologies , 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 Duration00:00:30

30s
Add Amount15 µL ofReagentElution Buffer (EB)Oxford Nanopore Technologies , spin down and resuspend, incubate at room temp for Duration00:10:00

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 Concentration1.2 ng/µL , we diluted Amount3 µL DNA: Amount2 µL ReagentElution Buffer (EB)Oxford Nanopore Technologies
·When the concentration of the DNA library was Concentration1.4 ng/µL , we diluted Amount2.9 µL DNA: Amount2.2 µL ReagentElution Buffer (EB)Oxford Nanopore Technologies





Setting Nanopore Parameters
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
10m
Priming buffer: mix Amount117 µL of ReagentFB (Flush Buffer)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 and Amount3 µL of ReagentFLT (Flush Tether)Oxford Nanopore TechnologiesCatalog #EXP-FLP002 in a tube





Mix Amount15 µL of Amount10 µL ReagentSequencing Buffer II (SBII)Oxford Nanopore Technologies of ReagentLoading Beads II (LBII)Oxford Nanopore Technologies and Amount5 µL of DNA library in a separate tube

.Insert Amount120 µL of the priming buffer into the flow cell

Insert Amount30 µL of prepped DNA library into flow cell

Demultiplexing
10m
Demultiplexing
Note
Full description of these procedures and the different options can be found here: https://github.com/asrivathsan/ONTbarcoder


Creating Demultiplexing file
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

Download Example demultiplexing text file.txtExample demultiplexing text file.txt

Merge resulting Fastq files

Note

Run ONTBarcoder https://github.com/asrivathsan/ONTbarcoder using default parameters.
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-x
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-x