TITLE: WORKFLOW FOR THE NUCLEIC ACID  BASED IDENTIFICATION OF INSECTS USING WHOLE GENOME AMPLIFICATION AND  NANOPORE SEQUENCING - Monarch®
AUTHORS: Jürg E Frey, Beatrice Frey, Daniel Frei, Morgan Gueuning, Simon Blaser, Andreas Bühlmann

[DESCRIPTION]

 


BACKGROUND
World-wide trade with plant material has dramatically increased over the past decades, and with it has  the risk for accidental introduction of potential plant pests and diseases. Rapid and accurate nucleic acid based identification of such quarantine organisms has become an important tool to minimize their  dispersal. 
Nucleic acid based identification exploits genetic diversity. A basic tenet holds that species generally do not interbreed and hence, the level of genetic differentiation within species is generally lower than between species. For insects, this pattern of genetic diversity is being used by DNA barcoding with  great success. An approximately 600 base pairs long fragment in the first half of the mitochondrially encoded cytochrome c oxidase I gene (COI) is used as a reference sequence to identify insects at the species level. So far, reference sequences for ca. 231’000 different insect species are deposited on the publicly available “Barcoding for Life Database” BOLD (http://www.barcodinglife.org/; by June 2021). Another important database especially targeting phytosanitary purposes with DNA barcodes of vouchered reference specimen is the EPPO Q-Bank (European Plant Protection Organization, https://qbank.eppo.int/). 

The methodology of DNA barcoding generally relies on PCR amplification of the diagnostic COI gene fragment using a pair of primers for which the exact DNA sequence must be known. However, this information is not always available, for example in the case of so far undescribed species or in cases, where genetic variation within species affected primer sites. Furthermore, although the COI marker sequence shows an impressive degree of among species differentiation, this is not true for all species and hence, a number of important pest species cannot be differentiated based on this marker alone. 

An ideal method for species identification should therefore obtain information of the best discriminating genetic region or of several genetic regions. Here, we describe a state-of-the-art method to achieve  this task.

PURPOSE
The purpose of this workflow is to provide a generic method for genetic identification of potential insect quarantine species and of other especially dangerous pest species in support of the Swiss Federal Plant Protection Service. The method is marker independent and may be used with reference databases of any genetic fragment. It is based on whole genome amplification, followed by single strand nanopore sequencing and DNA barcoding based identification.

[BEFORE_START]
All protocol steps using commercial kits generally follow exactly the recommendations of the suppliers, omitting some of the more detailed comments. Deviations from the supplier’s protocols are clearly indicated.

This workflow performed successfully with tissue amounts corresponding to a single adult Thrips to pieces of max. 2 mg of, e.g., Tephritid larvae. If the weight of your insect sample is less than ca. 2 mg (e.g., an adult thrips or a small Drosophila species such as Drosophila suzukii) then use the entire sample. If the sample is larger (such as a Tephritid larva) cut off a small tissue sample of no more than 2 mg.
 


[GUIDELINES]
SCOPE
This method is suitable for the qualitative identification of DNA (deoxyribonucleic acid) or cDNA (reverse-transcribed DNA of RNA of, e.g., viruses) of potentially all organisms. It has been tested against a broad taxonomic range of pest species. The workflow is designed to work with fresh, ethanol (EtOH; preferably 70%) preserved and frozen samples. The workflow presented here is established for insect species identification, but it was successfully applied to the identification of fungi and bacteria using the proper reference databases.

DEFINITIONS & ABBREVIATION
DNA: Deoxyribonucleic acid  
PCR: Polymerase Chain Reaction  
SOP: Standard Operating Procedure  
UV: Ultraviolet  
WGA: Whole Genome Amplification 
CO1/COI: Mitochondrial cytochrome c oxidase 1 gene 
Bp: Base pairs

PRINCIPLE OF THE METHOD
The workflow starts with nucleic acid extraction (DNA and/or RNA followed by cDNA production), followed by a WGA step of the extracted DNA/cDNA, then a clean-up step before producing the nanopore sequencing library which finally is loaded on the MinION nanopore sequencing device. 

Several commercial kits were successfully used for nucleic acid extraction (mentioned in Section 'Materials') yet the best results for our workflow, both in terms of the number of active nanopores as well as the time to sequencing 200k reads, was obtained if a clean-up step was added after the extraction step. Here, we describe a workflow based on NEB's Monarch line for DNA extraction (New England Biolabs, Monarch Genomic DNA Purification Kit (Bioconcept AG, Allschwil, Switzerland, Order Nr. T3010S) using the provider's 'Protocol for Extraction and Purification of Genomic DNA from Tissues', followed by a reaction cleanup step using the Qiagen DNeasy PowerClean CleanUp Kit (Qiagen Instruments AG, Hombrechtikon, Switzerland, Order Nr. 12877-50).

The methods chosen for this workflow aim at flexibility of input material (gDNA, cDNA) and optimal output to enable multiple use of single flowcells. The workflow should in principle enable at least 10 individual runs of ca. 2-4 hours data collection on a single MinION flowcell. 

The resulting sequence data, ideally > 200’000 reads per individual sample, are loaded into the software ‘Geneious Prime’ and analyzed by a custom-made workflow using the proper reference library (see section "Materials"). 

7 Raw Data Processing and Analysis

7.1 Primary Data Acquisition and Basecalling

Primary data acquisition is performed with the Oxford Nanopore Technologies (ONT) data acquisition software MinKNOW GUI v.4.2.8, the operating software that drives nanopore sequencing devices. Basecalling of the raw nanopore sequencing read data is required to generate the fastq sequence data files needed for further analysis. Basecalling is a time-consuming process and hence it is beneficial to use graphic processing units (GPU) to support this process. To enable use of GPU for basecalling generally requires access to a Linux based operating system with decent memory and storage capacity. We use a Dell Precision 7920 Tower XCTO Base with 256GB RAM and a Nvidia Quadro RTX6000, 24 GB graphic card running Ubuntu 18.04. We use the software Guppy v. 4.5.4 for basecalling using GPU with a parameter set established by Miles Benton (https://gist.github.com/sirselim/2ebe2807112fae93809aa18f096dbb94#file-basecalling_notes-md) to be used from a terminal window: 
/guppy_basecaller --disable_pings --input_path /{location_of_data_folder}/ --save_path /{location_of_save_data_folder}/ -c dna_r9.4.1_450bps_hac.cfg -x 'auto' --recursive --num_callers 4 -- gpu_runners_per_device 8 --chunks_per_runner 1024 --chunk_size 1000 

The process generates fastq data files and places all reads with a minimum quality into a folder named “passed”.

7.2 Data analysis

To store and analyze the basecalled fastq nanopore read sequences obtained by sequencers from Oxford Nanopore Technologies (MinION, PromethION, GridION, Flongle) we use the software Geneious Prime v.21.1.1 or newer.

We developed an automated workflow that combines several steps including mapping reads to a reference database (in our case containing ca. 600 base pairs 5’ of the mitochondrial cytochrome oxidase I gene of insects, downloaded from the ‘Barcoding of Life Database’ BOLD), establishing a consensus sequence and running a BLAST search on a local copy of the GenBank database. The BLAST results which generally enable species identification of the sample are stored in a sub-folder.

Open the Geneious Prime software by clicking on the corresponding icon 
In the left panel, go to your working folder and right-click to generate a new folder for storage and analysis of your new nanopore data files. We recommend to use a new folder for each run and within that folder a sub-folder for each barcoded sample. The nanopore fastq data files are stored in a folder on the computer system running the software ‘MinKNOW’ (which enables performing nanopore sequencing on the MinION as mentioned above) with a name similar to ‘passed’ and have the extension ‘.fastq’. For each barcoded sample, the corresponding fastq files have to be copied into the newly established data sub-folder in Geneious, for example, via drag and drop. 
Select the desired number of read packets (by default, 4000 reads per packet), ideally ca. 200’000 in total, by activating them in the right-hand window, then select “Workflows – LB_MM2_PcRefSeq_NrSeq_ret30_210212” from the Geneious Prime menu. If that workflow (incl. in Appendix) does not appear on the menu you have to import it first.
In the new pop-up window, select the proper reference database – default is the custom made 60k entry database (RefDB_BOLD59kGBCocc755_60625Seq_211213.fasta; incl. in Appendix) extracted from BOLD and modified to exclude duplicates and to maintain a 97% minimal distance between branches. Upon confirming with ‘ok’ the workflow will perform the following steps using the parameters indicated below: 

    a) Mapper: Minimap2 v.2.17 with the following parameters: Dissolve contigs and re-assemble selected; reference     sequence: RefDB_BOLD59kGBCocc755_60625Seq_211213; data type: Oxford Nanopore; include secondary alignments: maximum secondary aligments per read: 5; minimum secondary to primary alignment score ratio: 0.8; no trimming (remove existing trim regions from sequences); and under advanced options, an additional command line option: -t 8. Also, in the results panel, select ‘Save assemply report’, ‘Save in sub-folder’ and ‘Save contigs’.
     b) Sort Documents: Field to sort by: % of Ref Seq; select ‘Reverse Sort’ and ‘After sorting, only keep at most 30 documents.
    c) Mask Alignment: Eliminates alignment columns with <35% entries; in the Options panel, select ‘Expose no options; in the Results panel, select ‘Save a copy with sites stripped; in the What to mask or strip panel, select ‘Sites containing: Gaps (%) 35%; in the All Operation Options panel, select ‘Don’t append ‘Stripped’ to sequence names. 
    d) Save Documents: Select ‘Save these documents as output from workflow’, ‘Save in subfolder called: {Folder Name}; ‘Select these documents when the operation completes, and ‘Continue’. 
    e) Generate Consensus Sequence: Establishes a consensus sequence with a 0% majority threshold. Uses the following parameters: In the Options panel, select ‘Expose no options; in the All Operation Options panel, select ‘Threshold: 0% - Majority’, ‘Ignore Gaps’, ‘Assign Quality Total’, ‘Trim to reference sequence’, and ‘Append text to name of alignment consensus sequence’. 
    f) Save Documents: Select ‘Save these documents as output from workflow’, ‘Save in subfolder called: {Folder Name}; ‘Select these documents when the operation completes, and ‘Continue’.
    g) BLAST: Performs a BLAST search for the ‘contig consensus sequence ‘ on a local GenBank database copy using the program ‘Megablast’, places results into a ‘Hit table’, retrieving ‘Matching regions with annotations’, and saves the top 10 best hits in a new folder. Uses the following parameters: In the Options panel, select ‘Expose no options; in the All Operation Options panel, select ‘Nucleotide Query Option’, Database ‘Nucleotide collection (nr/nt), Program: ‘Megablast’, Results: ‘Hit table’, Retrieve ‘Matching region with annotations’, Macimum Hits ‘10’. Also selected is ‘Low Complexity Filter’ and ‘Mask for lookup table’, with other parameters being default values. 
    h) Save Documents: Select ‘Save these documents as output from workflow’, ‘Save in subfolder called: {Folder Name}; and ‘Branch from 2 Operations Ago’. 
    i) Sort Documents: In the Options panel, select ‘Expose no options; In the Options panel, select Field to sort by: ‘Bit-Score’, and select ‘After sorting, only keep at most 30 documents.

The BLAST results in the new folder are sorted according to the highest Bit-Score for the hit which in most cases will present the highest % Pairwise Identity and the lowest probability value (E-Value) on top of the list. In addition, Geneious Prime adds other information for proper qualification of the BLAST hits. 

Generally, the results obtained with this workflow provide ‘% Pairwise Identity’ hits of >99% and hence strong evidence for an unambiguous species identification of the sample for which the consensus sequence was established.


7.3 Additional Resources

Geneious Prime: Training videos and other resources for all steps outlined above for the software Geneious Prime are available on the Geneious homepage: Resources | Geneious Prime 
Barcoding of Life Database: The Barcode of life systems page (http://www.boldsystems.org/) is an excellent resource for barcoding based identifications and it allows searching their database with the proper marker sequence (e.g., COI for insects and ITS for fungi; see detailed instructions below).


8 Barcode-based identification on the NCBI GenBank Database 

As an alternative to the identification using local BLAST implemented in the Geneious Prime workflow, the consensus sequence may also be BLASTed directly on the GenBank Database of NCBI:

Open the BLAST homepage of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and choose the web BLAST option ‘Nucleotide BLAST’. 
Paste your consensus sequence into the window ‘Enter Query Sequence’ 
Check that the following parameters are chosen: a. Under ‘Choose Search Set’, ‘Database’: Choose ‘Standard’, and ‘Nucleotide collection (nr/nt)’ in the dropdown menu; leave the other fields empty. b. Under ‘Program selection’, ‘Optimize for’: Choose ‘Highly similar sequences (megablast) c. Select ‘Show results in a new window’ before you select ‘BLAST’ at the bottom left of the web page d. It may take a short while before GenBank returns the BLAST results in a new window: 
Criteria for correct species allocation are, among others: a. The top >10 entries are the same species name, however, there may be synonymous names for many insects. b. The identity of the query sequence with the GenBank entry (column ‘Ident’) is >97% c. The coverage region of the alignment between your consensus sequence and the GenBank entry is >80% (at least 300bp) d. The E-Value is very close to 0
 


9 Barcode-based identification on the Barcode of Life Database

In order to identify what species your consensus, full-length COI sequence originates from it is necessary to utilize reference databases. One example for such reference database is the Barcode of Life Database (BOLD). This database includes a comprehensive set of COI sequence data that has been collected by individuals and organizations across the globe and is constantly being updated with new data. 

Start by navigating to the BOLD Systems webpage (http://www.boldsystems.org/) and select the “Identification” tab at the top of the webpage. 
Using the default settings, select the “Animal Identification (COI)” tab (for arthropod identification) and the “Species Level Barcode Records” database. Paste the consensus sequence obtained from the sample into the search box at the bottom of the page. 
The browser will eventually update and return the results of the search, revealing the records contained in the database that yield the closest match in terms of sequence similarity. The result screen contains a lot of information that may be explored to establish a confident identification for the query sequence. Often the search results will all originate from a single species allowing an unambiguous identification to be made for the sample. In some cases, two or more species are returned, prompting BOLD to display the message “A species match could not be made, the queried specimen is likely to be one of the following”. This may, for example, happen when highly similar reference sequences were entered with different names (synonyms) for the same species.


10 Barcode-based identification on the EPPO-Q-Bank Database 

Open the EPPO-Q-Bank homepage (https://qbank.eppo.int/blast?db=arthropods), check that ‘Arthropods’ are selected and paste your consensus sequence into the window ‘Paste sequence to align:’ and select ‘Start alignment’ 
EPPO-Q-Bank will return its results in the lower part of the same window: 
The percent identity of your query sequence to the best match entries in the EPPO-Q-Bank Database is indicated in the column ‘Similarity %’, the percent overlap of both sequences in the column ‘Overlap %’. If both values are close to 100% then the species allocation is reliable.
 


[STEPS]
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
1. Materials: 

Disrupt tissue samples on the Retsch Mixer Mill TissueLyser II (Qiagen), using the Monarch® Genomic DNA Purification Kit (New England Biolabs NEB #T3010) and Qiagen Collection Microtubes (racked) and Collection Microtube Caps (cat. nos. 19560 and 19566 respectively).
  
  

Notes before Starting: 
Add ethanol (≥ 95 %) to the Monarch gDNA Wash Buffer concentrate as indicated on the bottle label. 
Set a thermal mixer (e.g. ThermoMixer® or similar device), or a heating block to 56 °C for sample lysis. 
Set a heating block to 60 °C. Preheat the appropriate volume of elution buffer to 60 °C (35 µL–100 µL per sample). Confirm the temperature, as temperatures are often lower than indicated on the device.

SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
2. All samples should be stored frozen at -20 °C or stored in 70 % (few days at -20 Room temperature or at 4 °C in the refrigerator) until processed. Samples can be stored frozen indefinitely. Use sterile dissection equipment where appropriate. 
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
3. Add 200 µL and 10 µL to each sample.
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
4. Add one stainless steel ball 3 mm per sample.
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
5. Place max. 2 mg into a Collection Microtube (Qiagen).
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
6. Disrupt tissue on the TissueLyser II for 2x3 min at 25 Hz, turning plate after the first period. Briefly centrifuge once done.
 

SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
7. Incubate at 56 °C for 30 min in a thermal mixer with agitation at full speed (1400 rpm) .
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
8. Centrifuge for 3 min at maximum speed (> 12.000 x g) to pellet debris. Transfer the supernatant to a fresh microfuge tube.
SECTION: 1 Sample preparation and DNA Extraction - 1.1 Tissue Disruption
9. Add 3 µL to the lysate, vortex thoroughly and incubate for a minimum of 5 min at 56 °C with agitation at full speed.

SECTION: 1.2 DNA Binding and Elution
10. Add 400 µL to the sample and mix thoroughly by pulse-vortexing for 5 s-10 s.
SECTION: 1.2 DNA Binding and Elution
11. Transfer the lysate/binding buffer mix (~600 µL) to a gDNA Purification Column pre-inserted into a collection tube, without touching the upper column area. Close the cap and centrifuge: first for 3 min at 1.000 x g to bind gDNA (no need to empty the collection tubes or remove from centrifuge) and then for 1 min at maximum speed (> 12.000 x g) to clear the membrane. Discard the flow-through and the collection tube.
SECTION: 1.2 DNA Binding and Elution
12. Transfer column to a new collection tube and add 500 µL. Close the cap and invert a few times, so that the wash buffer reaches the cap. Centrifuge immediately for 1 min at maximum speed (12.000 x g), and discard the flow through.
SECTION: 1.2 DNA Binding and Elution
13. Reinsert the column into the collection tube. Add 500 µL and close the cap. Centrifuge immediately for 1 min at maximum speed (>12.000 x g), then discard the collection tube and flow through.
SECTION: 1.2 DNA Binding and Elution
14. Place the gDNA Purification Column in a DNase-free 1.5 ml microfuge tube (not included). Add 50 µL, close the cap and incubate at Room temperature for 1 min.
SECTION: 1.2 DNA Binding and Elution
15. Centrifuge for 1 min at maximum speed (> 12.000 x g) to elute the gDNA.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
16. Materials: 

The reaction cleanup is performed using the DNeasy® PowerClean® Cleanup Kit (Qiagen order nr. 12877-50) according to the manufacturer’s recommendations.
  

Notes before Starting: 
Shake to mix solution SB. 
If Solution SL has precipitated, heat at 60 °C, gently inverting the tube periodically until the precipitate has dissolved. Solution SL may be used while still warm.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
17.  Add 100 µL to the 50 µL.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
18. Transfer 150 µL to a clean 2 ml collection tube (provided).
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
19. Add 70 µL to the DNA. Gently invert 5 times.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
20. Add 20 µL and invert 5 times.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
21. Add 85 µL and invert 5 times. Incubate at 4 °C (e.g., in a refrigerator) for 5 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
22. Centrifuge at 10000 x g, 1 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
23. Transfer supernatant to clean 2 ml collection tube (provided), do not disturb pellet.

SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
24. Add 70 µL  and invert 5 times. Incubate at 4 °C for 5 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
25. Centrifuge at 10000 x g, 1 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
26. Transfer supernatant to clean 2 ml collection tube (provided), do not disturb pellet.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
27. Add 800 µL and vortex for 5 s.

SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
28. Load600 µL onto an MB Spin Column and centrifuge at 10000 x g, 1 min. Discard flow through.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
29. Add the remaining 600 µL to the MB Spin Column and centrifuge at 10000 x g, 1 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
30. Add 500 µL to the MB Spin Column and centrifuge at 10000 x g, 30 s. Discard flow through.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
31. Centrifuge the MB Spin Column at 13000 x g, 1 min.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
32. Carefully place the MB Spin Column in new 2 ml collection tube (provided). Avoid splashing any Solution CB onto the MB Spin Column.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
33. Add 50 µL to the center of the white filter membrane. Incubate for 1 min at 4 Room temperature.
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
34. Centrifuge at 10000 x g, 30 s. 
SECTION: 2 Reaction Cleanup - 2.1 Cleanup of eluted DNA
35. Discard the MB Spin Column. Continue with WGA or store cleaned DNA frozen at -20 °C.
SECTION: 2.2 DNA quantification
36. DNA extracted with a commercial kit or after clean-up may be quantified to assess the extraction process and enable normalisation of DNA concentration. One common method is to use a Qubit 3 fluorometer or, alternatively, a Nanodrop ND 1000 spectrophotometer. DNA should be diluted to 10-50ng/μl using DNA-free water. Negative controls should read ~0 ng/μl.

Controls:  
A negative extraction control (with no tissue) should be run in parallel with all batches of sample extraction and quantified alongside all tissue extractions
SECTION: 3 Whole Genome Amplification (WGA)
37. Materials:  

We use the GenomePlex® Complete Whole Genome Amplification Kit WGA2 (Sigma-Aldrich  Chemie GmbH, Buchs, Switzerland; Product code WGA2-50RXN).
  

Procedure for whole genome amplification using the Sigma GenomePlex® Complete Whole Genome  Amplification Kit WGA2: 
SECTION: 3.1 WGA Step 1: Fragmentation
38. Run Thermocycler program (Program: incubation at 95 °C, runs 30 min).
(To assure the Thermocycler is ready when needed.)
SECTION: 3.1 WGA Step 1: Fragmentation
39. Use DNA/cDNA sample: Transfer 10 µL of section 2.2/step 19 into new 8-Strip Microtubes.
SECTION: 3.1 WGA Step 1: Fragmentation
40. Add 1 µL to each DNA tube of previous step.
SECTION: 3.1 WGA Step 1: Fragmentation
41. Heat for 4 min @ 95 °C in Thermocycler. Immediately cool on ice.
 

SECTION: 3.2 WGA Step 2: Library Preparation
42. Add 2 µL to DNA of  previous step.
SECTION: 3.2 WGA Step 2: Library Preparation
43. Add 1 µL to DNA of previous step. Vortex and centrifuge.
SECTION: 3.2 WGA Step 2: Library Preparation
44. Heat for 2 min @ 95 °C in Thermocycler. Immediately cool on ice.
SECTION: 3.2 WGA Step 2: Library Preparation
45. Add 1 µL to DNA of previous step. Vortex and centrifuge.

SECTION: 3.2 WGA Step 2: Library Preparation
46. Run Thermocycler program with WGA Library Prep Rxn.

Program: 
incubation at 16 °C, runs 20 min; 
incubation at 24 °C, runs 20 min; 
incubation at 37 °C, runs 20 min; 
incubation at 75 °C, runs 5 min; 
cool to and hold at 4 °C
SECTION: 3.3 WGA Step 3: Amplification
47. Add 48 µL to each reaction tube of previous step (WGA Library Prep Rxn).
SECTION: 3.3 WGA Step 3: Amplification
48. Add 7.5 µL to each reaction tube of previous step.
SECTION: 3.3 WGA Step 3: Amplification
49. Add 5 µL to each reaction tube of previous step. Vortex and centrifuge.
SECTION: 3.3 WGA Step 3: Amplification
50. Run Thermocycler program.

Program:
Initial incubation: 3 min at 95 °C;
17 cycles of 15 s at 94 °C; 5 min at 65 °C;
cool to and hold 4 °C

Store short term 4 °C, long term -20 °C.
SECTION: 3.3 WGA Step 3: Amplification
51. OPTIONAL: Check on gel: Run 5 µL on 1.4% TBE gel, 4 µL, 4 µL at 70 V/cm for 30 min.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
52. Prepare MinElute column setup on 2ml collection tube.



SECTION: 3.4 WGA Step 4: Reaction Cleanup
53. Add 300 µL to 75 µL.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
54. Load 375 µL to column setup.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
55. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
56. Add 750 µL to column setup.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
57. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
58. Centrifuge the sample of last step for 2 min. 
Place MinElute column in NEW 1.5ml  Eppendorf tube.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
59. Add 10 µL to centre of MinElute Column.
 

SECTION: 3.4 WGA Step 4: Reaction Cleanup
60. Incubate for 1 min @ -20 Room temperature.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
61. Centrifuge for 1 min.
 

SECTION: 4 WGA Product Check by Gel Electrophoresis
62. Gel electrophoresis of DNA in an agarose gel is a standard technique in molecular biology, but equipment, reagents, staining and visualization varies considerably between laboratories, and according to  local health & safety controls. Therefore, this SOP suggests general conditions that need to be  adapted to each laboratory. 
SECTION: 4.1 Make a 1.2% TBE agarose gel (1xTBE pH: 9.0) containing 0.0001% Ethidium Bromide*)
63. Place 80 mL + 1 g in a 500ml Erlenmeyer flask.
SECTION: 4.1 Make a 1.2% TBE agarose gel (1xTBE pH: 9.0) containing 0.0001% Ethidium Bromide*)
64. Heat in microwave at max intensity for 2 min with intermittent interruption for shaking (take care not to overheat).

SECTION: 4.1 Make a 1.2% TBE agarose gel (1xTBE pH: 9.0) containing 0.0001% Ethidium Bromide*)
65. Add 8 µL and cast the gel.
 


SECTION: 4.1 Make a 1.2% TBE agarose gel (1xTBE pH: 9.0) containing 0.0001% Ethidium Bromide*)
66. Wait 30 min at -20 Room temperature or store in the fridge at 4 °C.
SECTION: 4.2 Gel loading and running
67. Prepare Size Standard (e.g. Thermo Scientific™ GeneRuler DNA Ladder Mix, ready-to-use;  Order Nr. 10181070) and samples (whole genome amplification products) for gel loading  by mixing 3 µL with 3 µL to be  prepared as follows:

Loading Buffer Preparation for PCR Amplification Product Electrophoresis (10ml):



SECTION: 4.2 Gel loading and running
67.1. Add 1.5 g to 10 mL, adjust pH to pH 9.0.
SECTION: 4.2 Gel loading and running
67.2. Add 5 mg (adjust amount visually, may be too high).
SECTION: 4.2 Gel loading and running
68. Carefully pipet the 6 µL into individual wells of the gel, beginning with the Size Standard at the leftmost well.

SECTION: 4.2 Gel loading and running
69.  Run at 70V for approximately 30 min (depending on size of gel), ensuring the DNA does not run off the gel.

SECTION: 4.3 Visualization
70. Visualize your DNA fragments in UV light (with appropriate safety precautions); if the WGA reaction has been successful it shows as a smear of approximately 400 - 1000 base pairs in length. Your negative controls should not contain bands.
SECTION: 4.3 Visualization
71. 
 



SECTION: 4.4 Recording
72. Keep a permanent record of your gel (electronic and/or hard copy) as proof that the WGA reaction was successful and contaminant free.
SECTION: 5 Sequencing Library Preparation
73. 
 

Materials:  

The library for nanopore sequencing is produced with the Ligation Sequencing Kit SQK-LSK109 of Oxford Nanopore Technologies for sequencing on the flowcell type R.9.4.1 (flowcell ID: FLO-Min106D), following the manufacturer’s recommendations with some minor modifications.
  
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
74. Transfer 120 ng total DNA of section 3 into new 8-strip Microtubes. 
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
75. Add MH2O to total 54 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
76. Add 3.5 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
77. Add 3 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
78. Mix by flicking, spin down.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
79. RUN Thermocycler program.

Program: 
incubate 30 min at 20 °C/20 min at 65 °C,
transfer contents to 1.5ml Eppendorf tube.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
80. Add 60 µL (resuspended). Mix by flicking tube.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
81. Incubate for 10 min @ 4 Room temperature on HULA mixer.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
82. Spin and pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
83. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
84. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
85. On magnet, pipette off residual EtOH.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
86. Dry for 30 s.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
87. Resuspend in 61 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
88. Incubate for 5 min at Room temperature

SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
89. Pellet on magnet until clear.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
90. Collect 61 µL eluate. May store at 4 °C 5 min.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
91. OPTIONAL: Quantify 1 µL of product on QuBit. Note DNA concentration (ng/μl).

SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
92. Add 22.5 µL of eluted DNA of Endrepair product into new 1.5 ml Eppendorf tube; mix by pipetting. Use total 100 fmol-200 fmol (=ca. 35-60 ng).
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
93. Add 2.5 µL to each reaction tube of previous step. Note Barcode Numbers!
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
94. Add 25 µL to each reaction tube of previous step; mix by pipetting.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
95. Incubate for 10 min @4 Room temperature.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
96. Add 50 µL (resuspended). Mix by flicking.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
97. Incubate for 5 min @4 Room temperature on HULA mixer. Spin down.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
98. Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
99. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
100. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
101. Remove residual by spin on magnet. Pipette off residual EtOH.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
102. Dry for 30 s.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
103. Resuspend in 26 µL.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
104. Incubate for 2 min @4 Room temperature.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
105. Pellet on magnet until clear.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
106. Collect 26 µL and transfer to 1.5ml Eppendorf tube.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
107. MUST DO: Quantify 1 µL of product on QuBit. Note DNA concentration (ng/μl).
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
108. Pool equimolar amounts of each barcoded sample to 1.5ml Eppendorf tube (to 100-200 fmol total (=ca. 60 ng)). 
 

SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
109. Dilute single pooled Barcode ligation product to 65 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
110. Use 60 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
111. Add 25 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
112. Add 10 µL.

SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
113. Add 5 µL. Mix by flicking, spin down.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
114. Incubate for 10 min @4 Room temperature.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
115. Add 40 µL. Mix by flicking
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
116. Incubate for 15 min @ 4 Room temperature on HULA mixer. Spin down.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
117. Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
118. Wash beads with 250 µL. Wait 3 min on Magnet. Resuspend by flicking, pellet, remove supernatant.

SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
119. Wash beads with 250 µL. Wait 3 min on Magnet. Resuspend by flicking, pellet, remove supernatant.

SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
120. Remove residual by spin.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
121. Dry 30 s.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
122. Resuspend in 15 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
123. Incubate for 10 min @ 37 °C.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
124. Pellet on magnet until clear.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
125. Collect library (15 µL) from previous step into new Eppendorf tube.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
126. Quantify 1 µL on QuBit. Use appropriate amount for 16 ng of library for next step; dilute with EB buffer.
SECTION: 6 Nanopore Sequencing
127. Priming and Loading the Flowcell
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
128. Prepare Flowcell (perform QC on MinION). Record number of active pores.
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
129. Prepare the flow cell priming mix: Add 30 µL directly to Flush Buffer (FB) tube. Mix.
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
130. Load flow cell with 800 µL via priming port. Spot on closed!!!
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
131. Wait for 5 min.

SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
132. Prepare Library for loading: add 38 µL to 1.5ml Eppendorf tube.
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
133. Add 26 µL to 1.5ml Eppendorf tube (mixed immediately before use).
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
134. Add 12 µL from section 5.3 (step 125) to 1.5 ml Eppendorf tube.
 


SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
135. Load flow cell  with 200 µL via priming port. Spot on closed!!!

SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
136. Add 75 µL from step 134 via SpotON sample port. Add drop by drop!

SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
137. Close priming port, SpotOn port, perform sequencing on a MinION (Flongle, GridION, Promethion) using protocols.io method https://www.protocols.io/view/starting-a-minion-sequencing-run-using-minknow-7q6hmze; make sure to use flow cell type LSK109 and barcode kit EXP-NBD104 (option now available).
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
138. Prepare wash mix: Add 20 µL to 1.5ml Eppendorf tube.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
139. Add 380 µL to same 1.5ml Eppendorf tube. Vortex.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
140. Open inlet port, add 400 µL via priming port. Close priming port after loading (Spot on closed!!!).
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
141. Wait for 30 min @37 Room temperature.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
142. Add 500 µL via priming port. Close priming port after adding (Spot on closed!!!).
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
143. Remove spare contents in flow cell. Aspirate 1000 µL from empty flow cell via trash removal port top left. Spot on closed!!!
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
144. Store in fridge.
SECTION: 7 Raw Data Processing and Analysis
145. For Raw Data Processing and Analysis, please see section "Guidelines".