ONT DNA Barcoding Fungal Amplicons w/ MinION & Flongle

Stephen       Douglas Russell

Mar 17, 2023

Version 3

ONT DNA Barcoding Fungal Amplicons w/ MinION & Flongle V.3

DOI

dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v3

Stephen Douglas Russell¹

¹The Hoosier Mushroom Society

Stephen Douglas Russell

Biodiverse, Mycota Lab

DOI: dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v3

Collection Citation: Stephen Douglas Russell 2023. ONT DNA Barcoding Fungal Amplicons w/ MinION & Flongle. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v3Version created by Stephen Douglas Russell

License: This is an open access collection 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 collection and it's working

Created: March 12, 2023

Last Modified: March 17, 2023

Collection Integer ID: 78626

Keywords: dna, sequencing, MinION, nanopore, oxford nanopore technologies, ONT, extraction, amplification, PCR, master mix, primers, NGSpeciesID, guppy, minibar, linux, PopOS, flongle, 10.4.1, adapter ligation, dA tailing, basecalling, demultiplex,

Abstract

This collection of protocols outline a working methodology to DNA barcode fungal specimens. This process will work for dried tissue, fresh tissue, or with DNA template that has already gone through an alternative extraction protocol. It takes you from computer/software setup through DNA extraction, amplification, library preparation, and sequencing It also includes primary and secondary data analysis. Take your fungal samples from specimens to GenBank accessions with this protocol!

This particular workflow is designed for use with Flongle 10.4.1 flowcells and V14 Ligation chemistry.

Materials

Total equipment needs and cost:
PCR tube rack x10 (Amazon) : $33.78 (would be good to have at least 10)
Tweezers (flat, non-serrated tips - Ebay): $10.00
Teasing needle (Amazon): $12.35
Mini centrifuge (Ebay): $70.00
PCR tube rack 1.5mL (Amazon): $26.42
0.5 -10uL multichannel pipette (Amazon): $155.51 (may be able to find used cheaper on Ebay)
50 - 300uL multichannel pipette (Amazon): $172.89 (may be able to find used cheaper on Ebay)
Summit Professional Freezer -20C (Facebook Marketplace / 1 / 2 ): $150 (gotta get lucky)
Magnetic bead separator for 1.5mL eppi tubes (Ebay): $59.00
Tip disposal bucket
Gel electrophoresis system (miniPCR): $300
Heat block (Amazon): $179.99
Hula mixer (Ebay): $200.00 (optional)
Flongle Starter Pack: $1,460.00
MinION Mk1B Starter Pack: $1000.00
Thermocycler: $100 - $1600

Total upfront equipment cost: ~$3,200 - $3,700, excluding thermocycler and computerTotal upfront equipment cost: ~$3,200 Total upfront equipment cost: ~$3,200 - $3,700, excluding thermocycler and computer- $3,700, excluding thermocycler and computer
Eppendorf Mastercycler (used) - $1600 (can find less expensive models used, reasonable units in the $200-$400 range)
System 76 laptop - $4,000 (can probably find something that works for much cheaper...depending on your use case)

Consumables:
0.2mL PCR tubes (Amazon): $12.83
0.2 non-skirted 96-well PCR plates (USA Scientific/Amazon): $22.00/10 x3 = $66.00
8-strip PCR caps (USA Scientific): $11.00/125 strips x3 = $33.00
PCR Sealing Film (Amazon): $36.74
Eppendorf DNA LoBind 1.5mL tubes (USA Scientific): $31.75 per 250 tubes
15mL tubes (Amazon): $17.99
10ul-100uL Pipette (Amazon): $32.39
1000uL pipette (Amazon): $32.39
10uL filtered pipette tips (Amazon): $63.99
200uL filtered pipette tips (Amazon): $57.77
1000uL pipette tips (Amazon): $13.28
Alcohol swabs (Amazon): $5.49
Kimwipes (Amazon): $24.30
Fine-tip Sharpies (Amazon): $1.84
Eliminase (Ebay): $60.00

Total upfront consumable purchases: $400 - $500

Primers
ONT-tagged Forward Primers  $87.50
ONT-tagged Reverse Primers $396.16

Total upfront primer cost: ~$500

Reagents:
X-Amp DNA Reagent  (IBI Scientific): 50mL kit is $128.47. Cost per sample between $0.039 (15uL) - $0.064 (25uL)
PCR Master Mix (Empirical Bioscience): $206.90 shipped  ($10.34 per plate; $0.108 per reaction/sample)
Ethanol:  $56.18 per 1L
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S  $283.00 per 24 reactions
ReagentMolecular WaterIBI ScientificCatalog #IB42130  
ReagentHighPrep™ PCR Clean-up SystemMagBio Genomics Inc.Catalog #AC-60005 : $117.88 per 50 mL. $0.047 per rxn.
ReagentLigation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114  : $694.43 per 6 reactions (MinION) or 12 reactions (Flongle)
ReagentNEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S  : $361.00 per 20 reactions

Total upfront reagent costs:  ~$1,600

Extraction ongoing costs:
Total per sample: $0.071
Total per 96 samples: $6.88
Total per Flongle run (480 samples) : $34.40
Total per Flongle run (672 samples) : $48.16
Total per Flongle run (960 samples) : $68.88

PCR ongoing costs:
Total per sample: ~$0.10  ($796.54 / 10,000 rxns from Empirical Biosciences)
Total per Flongle run (480 samples) : ~$48.00
Total per Flongle run (672 samples) : ~$67.20
Total per Flongle run (960 samples) : ~$96.00

dA-tailing ongoing costs:
Total per Flongle run (1/2 rxns): $5.95
Total per MinION run: $11.85
Total per sample (Flongle: 480 samples): $0.012   
Total per sample (Flongle: 672 samples): $0.0089
Total per sample (Flongle: 960 samples): $0.0061

Ligation ongoing costs:
Total per Flongle run (1/2 rxns): $66.95
Total per MinION run: $133.84
Total per sample (Flongle: 480 samples): $0.139
Total per sample (Flongle: 960 samples): $0.07

Sequencing ongoing costs:
Flongle cells: $810 per 12 cells + $50 per shipment (likely want 3-4 shipments) = ~$100 per cell



Total upfront costs:
~$5700 - $6200 + thermocycler + compute

Total ongoing costs:
Per Flongle run: $255 per 480 samples = $0.53 per specimen
Per Flongle run: $288 per 672 samples = $0.43 per specimen
Per Flongle run: $338 per 960 samples = $0.35 per specimen