Jul 17, 2022
Version 1
ONT DNA Barcoding Fungal Amplicons w/ MinION & Flongle V.1
- 1The Hoosier Mushroom Society
Collection Citation: Stephen Douglas Russell 2022. ONT DNA Barcoding Fungal Amplicons w/ MinION & Flongle. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v1
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: June 09, 2022
Last Modified: July 17, 2022
Collection Integer ID: 64290
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, sequencing, and primary data analysis.
This particular workflow is designed for use with Flongle 9.4.1 flowcells and V12 Ligation chemistry.
Materials
Total equipment needs and cost:
Tweezers (flat, non-serrated tips - Ebay): $10.00
Tip disposal bucket
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 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:
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
NEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S $283.00 per 24 reactions
Molecular WaterIBI ScientificCatalog #IB42130
HighPrep™ PCR Clean-up SystemMagBio Genomics Inc.Catalog #AC-60005 : $117.88 per 50 mL. $0.047 per rxn.
Ligation Sequencing Kit (Q20 )Oxford Nanopore TechnologiesCatalog #SQK-LSK112 : $694.43 per 6 reactions
NEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S : $361.00 per 20 reactions
Total upfront reagent costs: ~$1,600
Extraction ongoing costs:
Total per sample: $0.093 - $0.118
Total per 96 samples: $8.93 - $11.33
Total per Flongle run (480 samples) : $44.65 - $56.65
Total per Flongle run (672 samples) : $62.50 - $79.30
PCR ongoing costs:
Total per sample: ~$0.225
Total per 96 samples: ~$22.50
Total per Flongle run (480 samples) : ~$110.00
Total per Flongle run (672 samples) : ~$150.00
dA-tailing ongoing costs:
Total per Flongle run (1/2 rxns): $5.95
Total per MinION run: $11.85
Total per 96 samples: $0.061
Total per sample (Flongle: 480 samples): $0.012
Total per sample (Flongle: 672 samples): $0.0089
Ligation ongoing costs:
Total per Flongle run (1/2 rxns): $66.95
Total per MinION run: $133.89
Total per 96 samples: $13.38
Total per sample (Flongle: 480 samples): $0.139
Total per sample (Flongle: 672 samples): $0.0996
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: $333 per 480 samples = $0.69 per specimen
Per Flongle run: $402 per 672 samples = $0.59 per specimen
Files
Protocol
Name
ONT Sequencing IT/Compute Pop!_OS 22.04 Setup
Version 1
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
Preparing ONT-tagged Primers and Master Mix for Fungal DNA Barcoding
Version 2
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT DNA Extraction for Fungal Barcoding
Version 1
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT Post-PCR Pooling & Purification for Fungal Barcoding
Version 2
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT dA-tailing for Fungal Barcoding
Version 2
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT Q20+ (V12) Adapter Ligation for Fungal DNA Barcoding
Version 2
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT Flongle Flowcell Loading with Q20+ (V12) Chemistry
Version 2
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society
Protocol
Name
ONT Basecalling, Demultiplexing, and Analysis for Fungal Barcodes
Version 1
Stephen Douglas RussellMycota Lab / The Hoosier Mushroom Society