TITLE: WORKFLOW FOR THE NUCLEIC ACID  BASED IDENTIFICATION OF INSECTS USING WHOLE GENOME AMPLIFICATION AND  NANOPORE SEQUENCING - KAWA
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.

[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. 

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 greater.

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 59k 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
1. All samples should be stored frozen at -20 °C or stored in 70 % (few days at Room temperature or  at 4 °C in the refrigerator) until processed. Samples can be stored frozen indefinitely. Use sterile dissection equipment where appropriate. As we are mostly using crude DNA extracts with or without further  cleanup for the WGA step there is a need to equilibrate tissue size with the amount of proteinase K buffer solution. The following table lists empirically established conditions:

 

SECTION: 2 DNA Extraction
2. Materials:  

The extraction is carried out with the proteinase K containing KAWA buffer (section 2.1 / step 3). We use Qiagen Proteinase.K, 20 mg/ml (QIAGEN AG, Basel, Switzerland; Product code 19131). 
  

Alternatively, a commercial kit may be used as mentioned in Section Materials, following the manufacturer’s  protocols. It is recommended that the manufacturer’s guidelines are checked each time kits are ordered to ensure any updates/changes made since development of this SOP are incorporated. 
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
3. Add 100 µL-200 µL *) in Collection Microtubes (Qiagen).

*) KAWA Buffer: 10 Milimolar (mM), 1 Milimolar (mM), 0.5 %, 50 μg/ml, pH 8.0.

Preparation of 100 ml KAWA Buffer:

 

Aliquot in 1.5 ml tubes. 

STORE AT -20 °C.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
4.  Add stainless steel ball 3 mm.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
5. Add tissue sample.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
6. Homogenize on the Retsch Mixer Mill (Qiagen) for 2x3 min at 25 Hz, turning plate after the first period, then centrifuge.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
7. Heat tubes at 95 °C for 20 min.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
8. Centrifuge, transfer in 0.5 ml lowbind Eppendorf tubes.
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
9. Store frozen at -20 °C (avoid freeze-thaw cycles, homogenates may be stored for several weeks at 4 °C).
SECTION: 2.1 Fast Extraction by Proteinase K Buffer (KAWA extraction)
10. 
 

SECTION: 2.2 Reaction cleanup
11. Materials:  

We use the MinElute Reaction Cleanup Kit of Qiagen (QIAGEN AG, Basel, Switzerland; Product code 28206). 
  

A cleanup step using the MinElute Reaction Cleanup Kit of Qiagen (QIAGEN AG, Basel, Switzerland; Product code 28206) is recommended after the proteinase K extraction as this seems to reduce the variance in the number of sequencing reads between individual libraries in multiplex runs. 

This step is not necessary if using a commercial nucleic acid extraction kit. However, a reverse transcription step is required if using an RNA extraction kit. For example, using the GenElute™ Total RNA Purification Kit from Sigma-Aldrich (Merck, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland; Product  code: RNB100), the following cDNA production kit was successfully used: LunaScript® RT SuperMix Kit (Bioconcept AG, Allschwil, Switzerland; Product code NEB E3010S). Also, if RNA is extracted it may be beneficial to omit the DNAse step to maximize the yield of nucleic acids.
  
  

Procedure for reaction cleanup using the Qiagen MinElute Reaction Cleanup Kit:
SECTION: 2.2 Reaction cleanup
12. Prepare MinElute column setup on 2ml collection tube.

SECTION: 2.2 Reaction cleanup
15. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 2.2 Reaction cleanup
16. Add 750 µL to column setup.
SECTION: 2.2 Reaction cleanup
17. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 2.2 Reaction cleanup
18. Centifuge the sample of last step for 2 min. 
Place MinElute column in NEW 1.5ml  Eppendorf tube.
SECTION: 2.2 Reaction cleanup
19. Add 12 µL to centre of MinElute Column.
 


SECTION: 2.2 Reaction cleanup
20. Incubate for 1 min @ -20 Room temperature.
SECTION: 2.2 Reaction cleanup
21. Centrifuge for 1 min.
 


SECTION: 2.3 DNA extract quantification
22. 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 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)
23. 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
24. 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
25. 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
26. Add 1 µL to each DNA tube of previous step.
SECTION: 3.1 WGA Step 1: Fragmentation
27. Heat for 4 min @ 95 °C in Thermocycler. Immediately cool on ice.
 

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

SECTION: 3.2 WGA Step 2: Library Preparation
32. 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
33. Add 48 µL to each reaction tube of previous step (WGA Library Prep Rxn).
SECTION: 3.3 WGA Step 3: Amplification
34. Add 7.5 µL to each reaction tube of previous step.
SECTION: 3.3 WGA Step 3: Amplification
35. Add 5 µL to each reaction tube of previous step. Vortex and centrifuge.
SECTION: 3.3 WGA Step 3: Amplification
36. 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
37. 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
38. Prepare MinElute column setup on 2ml collection tube.



SECTION: 3.4 WGA Step 4: Reaction Cleanup
41. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
42. Add 750 µL to column setup.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
43. Centifuge the sample of last step for 1 min. 
Discard flow-through, re-assemble.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
44. 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
45. Add 10 µL to centre of MinElute Column.
 

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

SECTION: 4 WGA Product Check by Gel Electrophoresis
48. 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*)
49. 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*)
50. 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*)
51. 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*)
52. Wait 30 min at -20 Room temperature or store in the fridge at 4 °C.
SECTION: 4.2 Gel loading and running
53. 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
53.1. Add 1.5 g to 10 mL, adjust pH to pH 9.0.
SECTION: 4.2 Gel loading and running
53.2. Add 5 mg (adjust amount visually, may be too high).
SECTION: 4.2 Gel loading and running
54. 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
55.  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
56. 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: 4.4 Recording
58. 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
59. 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
60. Transfer 120 ng total DNA of section 3 into new 8-strip Microtubes. 
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
61. Add MH2O to total 54 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
62. Add 3.5 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
63. Add 3 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
64. Mix by flicking, spin down.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
65. 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
66. Add 60 µL (resuspended). Mix by flicking tube.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
67. Incubate for 10 min @ 4 Room temperature on HULA mixer.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
68. Spin and pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
69. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
70. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
71. On magnet, pipette off residual EtOH.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
72. Dry for 30 s.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
73. Resuspend in 61 µL.
SECTION: 5.1 Library Preparation Step 1: DNA End-Prep
74. Incubate for 5 min at Room temperature

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

SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
78. 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
79. Add 2.5 µL to each reaction tube of previous step. Note Barcode Numbers!
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
80. Add 25 µL to each reaction tube of previous step; mix by pipetting.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
81. Incubate for 10 min @4 Room temperature.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
82. Add 50 µL (resuspended). Mix by flicking.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
83. Incubate for 5 min @4 Room temperature on HULA mixer. Spin down.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
84. Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
85. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
86. Wash beads on magnet with 200 µL. Pipette off supernatant, do not disturb pellet.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
87. Remove residual by spin on magnet. Pipette off residual EtOH.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
88. Dry for 30 s.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
89. Resuspend in 26 µL.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
90. Incubate for 2 min @4 Room temperature.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
91. Pellet on magnet until clear.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
92. Collect 26 µL and transfer to 1.5ml Eppendorf tube.
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
93. MUST DO: Quantify 1 µL of product on QuBit. Note DNA concentration (ng/μl).
SECTION: 5.2 Library Preparation Step 2: Native Barcode Ligation
94. 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
95. Dilute single pooled Barcode ligation product to 65 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
96. Use 60 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
97. Add 25 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
98. Add 10 µL.

SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
99. Add 5 µL. Mix by flicking, spin down.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
100. Incubate for 10 min @4 Room temperature.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
101. Add 40 µL. Mix by flicking
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
102. Incubate for 15 min @ 4 Room temperature on HULA mixer. Spin down.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
103. Pellet on magnet until clear. Pipette off supernatant, keep on magnet.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
104. 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
105. 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
106. Remove residual by spin.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
107. Dry 30 s.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
108. Resuspend in 15 µL.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
109. Incubate for 10 min @ 37 °C.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
110. Pellet on magnet until clear.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
111. Collect library (15 µL) from previous step into new Eppendorf tube.
SECTION: 5.3 Library Preparation Step 3: Adaptor Ligation and Clean-up
112. Quantify 1 µL on QuBit. Use appropriate amount for 16 ng of library for next step; dilute with EB buffer.
SECTION: 6 Nanopore Sequencing
113. Priming and Loading the Flowcell
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
114. Prepare Flowcell (perform QC on MinION). Record number of active pores.
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
115. 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
116. Load flow cell with 800 µL via priming port. Spot on closed!!!
SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
117. Wait for 5 min.

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


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

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

SECTION: 6.1 Nanopore Sequencing Step 1: Priming and Loading the Flowcell
123. 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
124. Prepare wash mix: Add 20 µL to 1.5ml Eppendorf tube.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
125. Add 380 µL to same 1.5ml Eppendorf tube. Vortex.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
126. 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
127. Wait for 30 min @37 Room temperature.
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
128. Add 500 µL via priming port. Close priming port after adding (Spot on closed!!!).
SECTION: 6.2 Nanopore Sequencing Step 2: Flow Cell Storage
129. 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
130. Store in fridge.
SECTION: 7 Raw Data Processing and Analysis
131. For Raw Data Processing and Analysis, please see section "Guidelines".
SECTION: 2.2 Reaction cleanup
13. Add 300 µL to 75 µL.
SECTION: 2.2 Reaction cleanup
14. Load 375 µL to column setup.
SECTION: 4.3 Visualization
57. 
 



SECTION: 3.4 WGA Step 4: Reaction Cleanup
39. Add 300 µL to 75 µL.
SECTION: 3.4 WGA Step 4: Reaction Cleanup
40. Load 375 µL to column setup.