TITLE: DNA Barcoding Protocol for Darwin Tree of Life at The Natural History Museum of London
AUTHORS: Jordan Beasley

[DESCRIPTION]
This protocol outlines the DNA barcoding process at the Natural History Museum of London (NHM) for the Darwin Tree of Life project (DToL). DNA barcoding is used as part of the species identification process and sample tracking to check that the genome sequence corresponds to the material that was sent.

[BEFORE_START]
The DToL sample team prepare submissions for barcoding by removing a small piece of tissue from specimens and plating them out into 96 well plates in 80% Ethanol (refer to taxon specific SOPs). This is the format the samples are provided in for the following barcoding protocols.

[STEPS]
SECTION: DNA Extraction
1. Spin down the sample plate and inspect to check no tissue/sample is stuck in the lids of the sealing caps. 
SECTION: DNA Extraction
2. Remove the caps and remove the ethanol. Use a single channel pipette for very small samples and take care to ensure the sample is not aspirated into the tip and removed with the ethanol. 
SECTION: DNA Extraction
3. Place the plate on a thermocycler with no lid or seal and with the thermocycler lid open to allow the remaining ethanol to evaporate. Set the thermocycler to run at 70 ⁰C for 10 minutes or longer if required until remaining ethanol has evaporated off. Take care when moving the plate onto and off of the thermocycler without lids- static can cause dry samples to pop out of the wells.
SECTION: DNA Extraction
4. Prepare a lysis mastermix using the KAPA Express Extract kit (Table 1).


 

SECTION: DNA Extraction
5. Aliquot 50µl lysis master mix into each well and seal with a clear PCR seal. (Note: lysis master mix volume can be reduced for very small samples)
SECTION: DNA Extraction
6. Spin down the plate and incubate on a thermocycler using the following program:

 

SECTION: Lysate dilution
7. Make 1:10 dilution of lysate, adding 2.5µl lysate to 22.5µl PCR grade water
NOTE: Lysate can be stored at -20⁰C until needed.
SECTION: PCR with indexed primers
8. Prepare the PCR mastermix using the KAPA Plant PCR kit as follows:

 

(NOTE: This PCR amplifies a 750bp COI amplicon using primers containing indexes for ONT sequencing. The plant PCR kit is used because it was found result in a higher success rate for samples containing inhibitors.)
Primer sequences can be found here: 
  

SECTION: PCR with indexed primers
9. Aliquot 13.6µl PCR mastermix into each well.
SECTION: PCR with indexed primers
10. Add 1.6µl indexed primer mix to each well.
SECTION: PCR with indexed primers
11. Add 4.8µl 1:10 diluted lysate to each well
SECTION: PCR with indexed primers
12. Seal the plate with PCR seal and mix and spin the plate.
SECTION: PCR with indexed primers
13. Place the plate on a thermocycler and run the following program:

1 cycle:
95°C for 3 mins
5 cycles:
95°C for 40s
45°C for 40s
72°C for 1 min
40 cycles:
95°C for 40s
65°C for 40s
72°C for 1 min
1 cycle:
72°C for 5 mins
4°C ∞

NOTE: Plate can be stored at -20°C until needed.
SECTION: Gel QC
14. Pour 500ml 1x TAE into a glass duran bottle. (This makes enough gel solution to make two large gels with 4 rows of combs with 50 teeth each to take 2 plates of samples each).

SECTION: Gel QC
15. Weigh out 5g of agarose powder and add to the duran bottle. 
SECTION: Gel QC
16. Close the lid tightly and mix the agarose powder into the TAE by swirling the bottle.
SECTION: Gel QC
17. Loosen the lid so that air can escape the bottle (so it doesn't explode) and heat the bottle in a microwave in short bursts checking between heating until the liquid starts to boil and the agarose has gone into solution and the liquid is completely clear.
SECTION: Gel QC
18. Allow the agarose to cool so that it is still molten but not hot. Tape off the ends of a large gel tray with masking/autoclave tape and place combs in the tray.
SECTION: Gel QC
19. Pour half of the agarose (~250ml) into the tray, making sure there are no bubbles present and allow to set firm.
SECTION: Gel QC
20. When completely cooled and set, remove the plastic combs and tape, place in a tank filled with enough 1x TAE buffer to cover the gel.
SECTION: Gel QC
21. Add Gel Red to loading dye in a 1:1000 ratio. Load 2µl PCR product mixed with 3µl loading dye/Gel Red mix to the gel and run alongside 1Kb hyper ladder at 80 volts for ~45 mins. Check bands of the expected size are present by visualising the gel in a UV transilluminator.
SECTION: Pooling and quantification
22. Pool 5µl PCR product for each sample in your pool (taking care that no two samples in the pool have the same indexes). 
SECTION: Pooling and quantification
23. Quantify the pool using Qubit, taking 3 x 1µl replicates from the pool and take the average ng/µl from the three replicates as the concentration.
  

SECTION: Library Preparation and sequencing
24. Prepare library for sequencing using the Nanopore Ligation Sequencing kit V14 as described in the attached protocol.   
The library prep takes 500ng DNA in 24.5µl volume as input. 
The resulting library is sequenced on a flongle (v10.4.1) for 24 hours using the super-accurate basecalling setting.
SECTION: Analysis
25. Currently, ONTBarcoder software is used to extract consensus barcodes from the raw nanopore data. First fastq files from the run are concatenated into one fastq file containing all of the reads from the run.
This file is loaded into ONTBarcoder as input along with a demultiplexing file containing the indexing information for each sample (see manual below for details).
  
ONTBarcoder can be downloaded here: https://github.com/asrivathsan/ONTbarcoder
SECTION: Analysis
26. The barcodes from ONTBarcoder output are then searched in BOLD for species identification. In this pipeline, a BOLD search tool called BOLDigger is used to query multiple barcodes and return the top 20 hits in BOLD. Please see BOLDigger documentation here: https://github.com/DominikBuchner/BOLDigger