TITLE: Non-destructively barcoding hundreds of freshwater macroinvertebrates with a MinION
AUTHORS: Elise  C. Knobloch, Ray C Schmidt

[DESCRIPTION]


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). 




 


[GUIDELINES]
These protocols are modified from those described in Srivathsan et al 2021. 
 


[STEPS]
2. 
SECTION: Specimen preparation and DNA extraction
2.1. 
SECTION: Specimen preparation and DNA extraction
2.2. 
SECTION: Specimen preparation and DNA extraction
2.3. 
SECTION: Specimen preparation and DNA extraction
2.4. 
1.7. Cover with TempPlate sealing foil or reusable TempPlate pressure-fit sealing mat
SECTION: DNA extraction
1.6. 
Place one rinsed and dried specimen in each well head-first
 


 


 


SECTION: DNA extraction
1.5. Put 10 µL    in each well of a 96-well plate 

 

SECTION: Specimen preparation
1.4. Place specimen/s on a paper towel to dry for about 5 min
 


SECTION: Specimen preparation
1.3. Remove specimen/s from water, and rinse again in the second dish for 5 min 

SECTION: Specimen preparation
1.2. Remove specimen/s from ethanol and place in one dish of ddH2O for10 min 

 

 

SECTION: Specimen preparation
1.1. Fill two Petri dishes with ddH2O
SECTION: Specimen preparation and DNA extraction
1. 
Specimen preparation and DNA extraction

1.8. Place the covered 96-well plate in the thermal cycler
 

1.8. 
1.9. Heat at  65 °C for 18 min ,98 °C  for 2 min 

1.10. After heating, remove the  from each well and transfer to a clean 96-well plate
 


SECTION: DNA Dilution
1.11. Put 10 µL  of ddH2O in each well of a 96-well plate

SECTION: DNA Dilution
1.12. Put 1 µL of DNA extraction in each coinciding well

 

SECTION: PCR Amplification
2. PCR Amplification
SECTION: PCR Setup
2.1. 
Combine ddH2O,  , and forward primer in a tube
 
       Per 20µL   Reaction        H20      7        2x Buffer-APEX   red      10        Primer F      0.5        Primer R      0.5-not in   mastermix        DNA Template      2-not in   mastermix   
 
 

   

SECTION: PCR Setup
2.2. Distribute 17.5 µL of master mix into each tube of a strip tube
 


SECTION: PCR Setup
2.3. Distribute DNA template and reverse primer into each tube
 

SECTION: PCR Setup
2.4. Thermal Cycler Protocol:94 °C for 1 min, then 94 °C for 30 s, 42 °C for 1 min, 72 °C for 90 s, go back to step 2 39x, 72 °C for 7 min, infinite hold at 10 °C

 

SECTION: Gel Electrophoresis
2.5. 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. 
 

SECTION: Pooling and Cleaning
3. Pooling and cleaning

SECTION: Pooling and Cleaning
3.1. Take 4 µL from each PCR-This will yield approximately 1.152 mL of PCR product

SECTION: Pooling and Cleaning
3.2. Take 250 µL from the pooled PCR

 

SECTION: Pooling and Cleaning
3.3. Add 125 µL of    and pipette up and down to mix (Followed manufacturer protocol but listed here). 

 

SECTION: Pooling and Cleaning
3.4. Let sit for 10 min at room temperature

SECTION: Pooling and Cleaning
3.5. Let sit on magnetic rack for 2 min
 


SECTION: Pooling and Cleaning
3.6. Remove supernatant, leaving about 5 µL on the bottom of the tube

SECTION: Pooling and Cleaning
3.7. Add 250 µL of freshly made 70% ethanol and pipette gently up and down

SECTION: Pooling and Cleaning
3.8. Let sit on rack for 30 s

SECTION: Pooling and Cleaning
3.9. Remove all ethanol
SECTION: Pooling and Cleaning
3.10. Add another 250 µL of 70% ethanol and let sit for 30 s

SECTION: Pooling and Cleaning
3.11. Remove all ethanol
SECTION: Pooling and Cleaning
3.12. Let dry for 5 min to10 min  with the cap open so the ethanol can evaporate 



SECTION: Pooling and Cleaning
3.13. Add 40 µL of ddH2O and elute the pellet (remove from the rack)

SECTION: Pooling and Cleaning
3.14. Let sit for 2 min

SECTION: Pooling and Cleaning
3.15. Put back on the rack and let pellet form
 

SECTION: Pooling and Cleaning
3.16. Remove liquid away from pellet-->this is our cleaned product
 

SECTION: Pooling and Cleaning
3.17. Run in a gel compared to uncleaned product to evaluate success 
 

SECTION: Pooling and Cleaning
3.18. Quantify cleaned PCR with 

 

Qubit Fluorometer (QuantasTM Fluorometer protocol listed below)
1. Mix 1 µL of DNA sample with 200 µL of   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


SECTION: Demultiplexing
8. Demultiplexing
 


SECTION: Loading flow cell
7. Loading flow cell
 

SECTION: Library preparation
4. Library preparation 


 



SECTION: Library preparation
4.1. Dilute DNA-->concentration of DNA should be 100-200fmol amplicon DNA 

 

 

SECTION: Library preparation
4.2. Combine in a strip-tube: 22.5 µL of diluted DNA, 3.5 µL of  , 1.5 µL of  , and 2.5 µL of H2O

SECTION: Library preparation
4.3. Heat at 20 °C for 5 min, 65 °C for 5 min

SECTION: Library preparation
4.4. Transfer mix into new 1.5 ml tube
SECTION: Library preparation
4.5. Vortex  

SECTION: Library preparation
4.6. Add 30 µL of   to the mixture

SECTION: Library preparation
4.7. Incubate on Hula mixer for 5 min

SECTION: Library preparation
4.8. Spin down, place on magnet until clear
SECTION: Library preparation
4.9. Keeping the tube on the magnet, pipette off supernatant 
SECTION: Library preparation
4.10. Add 100 µLof 70% ethanol (do not touch pellet) 

SECTION: Library preparation
4.11. Remove ethanol
SECTION: Library preparation
4.12. Add 100 µL of 70% ethanol, take off magnet rack and spin down, put back on magnetic rack

SECTION: Library preparation
4.13. Remove ethanol, allow to dry for about 30 s

SECTION: Library preparation
4.14. Add 30.5 µL of ddH2), remove from magnetic rack and resuspend pellet 

SECTION: Library preparation
4.15. Incubate off rack for 2 min

SECTION: Library preparation
4.16. Place back on magnetic rack until clear
SECTION: Library preparation
4.17. Remove liquid and put in clean tube
SECTION: Loading flow cell
7.1. Priming buffer: mix 117 µL of   and 3 µL of   in a tube





SECTION: Loading flow cell
7.2. Mix 15 µL of 10 µL   of   and 5 µL of DNA library in a separate tube

SECTION: Loading flow cell
7.3. .Insert 120 µL of the priming buffer into the flow cell

SECTION: Loading flow cell
7.4. Insert 30 µL of prepped DNA library into flow cell

SECTION: Library preparation: Ligation
5. Ligation
SECTION: Library preparation: Ligation
5.1. Spin down  and   and put on ice. Thaw and vortex   and put on ice, thaw and mix   (EB)and put on ice, thaw   (SFB), vortex, put on ice











SECTION: Library preparation: Ligation
5.2. Combine in a new tube in this order: 30 µL of DNA from previous step, 12.5 µL of LNB, 5 µL of  , and 2.5 µL of AMX

SECTION: Library preparation: Ligation
5.3. Flick and spin mixture, incubate at room temp for 10 min

SECTION: Library preparation: Ligation
5.4. Vortex  

SECTION: Library preparation: Ligation
5.5. Add 20 µL of   to mixture and flick tube

SECTION: Library preparation: Ligation
5.6. Incubate at room temp on Hula mixer for 5 min

SECTION: Library preparation: Ligation
5.7. Spin sample, put on magnetic rack, let pellet form, pipette of supernatant
SECTION: Library preparation: Ligation
5.8. Add 125 µL of  , flick tube to resuspend, spin down, return tube to magnetic rack, let pellet form, remove supernatant 

SECTION: Library preparation: Ligation
5.9. Add 125 µL of  , flick tube to resuspend, spin down, return tube to magnetic rack, let pellet form, remove supernatant 
 



SECTION: Library preparation: Ligation
5.10. Allow to dry for about 30 s

SECTION: Library preparation: Ligation
5.11. Add 15 µL of , spin down and resuspend, incubate at room temp for 10 min

SECTION: Library preparation: Ligation
5.12. Place on magnetic rack and allow pellet to form, remove liquid and transfer to a clean tube
 

SECTION: Library preparation: Ligation
5.13. Quantify 
 

SECTION: Library preparation: Ligation
5.14. Dilute so the concentration of the DNA library is 3-20fmol
 

SECTION: Creating Demultiplexing file
8.1. 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

 

SECTION: Setting Nanopore Parameters
6. Setting Nanopore Sequencing Parameters 
SECTION: Creating Demultiplexing file
8.2. Save as a .txt file 

  

SECTION: Creating Demultiplexing file
8.3. Merge resulting Fastq files 

 

SECTION: Creating Demultiplexing file
8.4. Run ONTBarcoder https://github.com/asrivathsan/ONTbarcoder using default parameters. 
SECTION: Setting Nanopore Parameters
6.1. 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.  
SECTION: Creating Demultiplexing file
8.5. View resulting CO1 sequences for further analyses.