TITLE: Native Barcoding (SQK-NBD114) gDNA for Adaptive Sampling using Oxford Nanopore Technologies
AUTHORS: claire.anderson, Hannah Macpherson, Emil Gustavsson, Jasmaine Lee, zhongbo.chen

[DESCRIPTION]
This protocol describes how to carry out native barcoding of genomic DNA (gDNA) using the Native Barcoding Kit 24 V14 (SQK-NBD114.24) for Adaptive Sampling using Oxford Nanopore Technologies (ONT). Adaptive Sampling is a method of real-time selection of DNA molecules for sequencing and rejection of DNA molecules that are not of interest by reversing the voltage across the pores.

[GUIDELINES]
Native Barcoding Kit 24 V14 (SQK-NBD114.24) should only be used in combination with R10.4.1 flow cells (FLO-PRO114M). Load 4-5 barcoded samples per flow cell. 

For adaptive sampling the following expansion packs are required:

Sequencing Auxiliary Vials V14 (EXP-AUX003)
Flow Cell Wash Kit (EXP-WSH004)

 

Input DNA recommendations:

Quality check DNA length, quantity and purity before starting. It is important that the input DNA meets the quantity and quality requirements (see ONT notes on input DNA document).

High molecular weight DNA may require shearing prior to adaptive sampling to minimise pore blocking and increase read length N50 (see ONT notes of DNA fragmentation). We perform mechanical DNA fragmentation using Diagenode Megaruptor‱ 3 DNA shearing kit (and DNAFluid+ kit for viscous samples) to ensure uniform fragment lengths of 12-15 kb.
Shorter fragments are preferentially sequenced compared to longer fragments in a pool, which can be minimised by pooling samples of a similar size so that an equimolar mass of each sample is sequenced. Four-five barcoded libraries may be loaded per flow cell.

6 µg gDNA is enough starting material for fragmentation and two side by side library preparations per sample. This should yield enough library for 3x library loads at the optimal molar quantity of 50 fmol (see ONT notes on adaptive sampling).

Third-party reagents

Oxford Nanopore Technologies have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested.

Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents

 

Single-use tubes format with higher EDTA concentration:
 
            
  Name 
  Acronym 
  Cap colour 
  No. of
  vials 
  Fill volume per
  vial (µl)
  
  Native Barcodes 
  NB01-24 
  Clear 
  24
  (one per barcode)
  20
  
  DNA Control Sample 
  DCS 
  Yellow 
  2
  35
  
  Native Adapter 
  NA 
  Green 
  1
  40
  
  Sequencing Buffer 
  SB 
  Red 
  1
  700
  
  Library Beads 
  LIB 
  Pink 
  1
  600
  
  Library Solution 
  LIS 
  White
  cap, pink label 
  1
  600
  
  Elution Buffer 
  EB 
  Black 
  1
  500
  
  AMPure XP Beads 
  AXP 
  Amber 
  4
  1,200
  
  Long Fragment Buffer 
  LFB 
  Orange 
  1
  1,800
  
  Short Fragment Buffer 
  SFB 
  Clear 
  1
  1,800
  
  EDTA† 
  EDTA 
  Clear 
  1
  700
  
  Flow Cell Flush 
  FCF 
  Blue 
  6
  1,170
  
  Flow Cell Tether 
  FCT 
  Purple 
  1
  200
  
  
 


Native barcode sequences:
 
        
  Component 
  Forward sequence 
  Reverse sequence
  
  NB01 
  CACAAAGACACCGACAACTTTCTT 
  AAGAAAGTTGTCGGTGTCTTTGTG
  
  NB02 
  ACAGACGACTACAAACGGAATCGA 
  TCGATTCCGTTTGTAGTCGTCTGT
  
  NB03 
  CCTGGTAACTGGGACACAAGACTC 
  GAGTCTTGTGTCCCAGTTACCAGG
  
  NB04 
  TAGGGAAACACGATAGAATCCGAA 
  TTCGGATTCTATCGTGTTTCCCTA
  
  NB05 
  AAGGTTACACAAACCCTGGACAAG 
  CTTGTCCAGGGTTTGTGTAACCTT
  
  NB06 
  GACTACTTTCTGCCTTTGCGAGAA 
  TTCTCGCAAAGGCAGAAAGTAGTC
  
  NB07 
  AAGGATTCATTCCCACGGTAACAC 
  GTGTTACCGTGGGAATGAATCCTT
  
  NB08 
  ACGTAACTTGGTTTGTTCCCTGAA 
  TTCAGGGAACAAACCAAGTTACGT
  
  NB09 
  AACCAAGACTCGCTGTGCCTAGTT 
  AACTAGGCACAGCGAGTCTTGGTT
  
  NB10 
  GAGAGGACAAAGGTTTCAACGCTT 
  AAGCGTTGAAACCTTTGTCCTCTC
  
  NB11 
  TCCATTCCCTCCGATAGATGAAAC 
  GTTTCATCTATCGGAGGGAATGGA
  
  NB12 
  TCCGATTCTGCTTCTTTCTACCTG 
  CAGGTAGAAAGAAGCAGAATCGGA
  
  NB13 
  AGAACGACTTCCATACTCGTGTGA 
  TCACACGAGTATGGAAGTCGTTCT
  
  NB14 
  AACGAGTCTCTTGGGACCCATAGA 
  TCTATGGGTCCCAAGAGACTCGTT
  
  NB15 
  AGGTCTACCTCGCTAACACCACTG 
  CAGTGGTGTTAGCGAGGTAGACCT
  
  NB16 
  CGTCAACTGACAGTGGTTCGTACT 
  AGTACGAACCACTGTCAGTTGACG
  
  NB17 
  ACCCTCCAGGAAAGTACCTCTGAT 
  ATCAGAGGTACTTTCCTGGAGGGT
  
  NB18 
  CCAAACCCAACAACCTAGATAGGC 
  GCCTATCTAGGTTGTTGGGTTTGG
  
  NB19 
  GTTCCTCGTGCAGTGTCAAGAGAT 
  ATCTCTTGACACTGCACGAGGAAC
  
  NB20 
  TTGCGTCCTGTTACGAGAACTCAT 
  ATGAGTTCTCGTAACAGGACGCAA
  
  NB21 
  GAGCCTCTCATTGTCCGTTCTCTA 
  TAGAGAACGGACAATGAGAGGCTC
  
  NB22 
  ACCACTGCCATGTATCAAAGTACG 
  CGTACTTTGATACATGGCAGTGGT
  
  NB23 
  CTTACTACCCAGTGAACCTCCTCG 
  CGAGGAGGTTCACTGGGTAGTAAG
  
  NB24 
  GCATAGTTCTGCATGATGGGTTAG 
  CTAACCCATCATGCAGAACTATGC
  

Designing an experiment with adaptive sampling

After considering factors like % of the genome to enrich for, read lengths and response time, the next step is to collate the input files needed for an adaptive sampling experiment. These are:
A genomic reference: this must be a FASTA or a pre-calculated minimap2 index
A .bed file with the genomic coordinates of the target region/s, including flanking "buffer" regions (see ONT notes on adaptive sampling)

Note that “Buffer” regions are flanking regions added to both sides of every single target described in the .bed file. These regions allow reads which begin with a sequence that does not initially map to the target region, but may extend into the target region, to be accepted. By accepting reads which map into these flanking regions, the number of accepted reads that hit our target is increased. ONT recommend setting the buffer size to the read-N10 of the library (this is point at which 10% of the bases sequenced are from reads that are this length or longer). Having a buffer size that exceeds the length of all reads present in the library will lead to reads being accepted and sequenced that never enter the target region.

Note that if you do not input a .bed file, then the entire FASTA/minimap2 index file will be used for enrichment or depletion.

PromethION 24 computer requirements for adaptive sampling

The PromethION 24 DAU device with 2 x Nvidia Quadro GV100 GPUs allows ≤3 positions/flow cells to run with real-time high accuracy basecalling required for adaptive sampling. Exceeding these recommendations may lead to an increased decision time and less enrichment.

Check your PromethION flow cell

Oxford Nanopore Technologies will replace PromethION flow cell/s with <5000 pores when the storage recommendations have been followed, if the check is performed within three months of purchase and the result is reported within two days of performing the flow cell check.

Troubleshooting

Below is a list of the most commonly encountered issues, with some suggested causes and solutions. Also consult the FAQ section available on the Nanopore Community Support section.

For adaptive sampling-specific troubleshooting, refer to the adaptive-sampling-ADS_S1016_v1_revJ_12Nov2020-any document and ONT notes on adaptive sampling.

If issues persists, contact ONT Technical Support (support@nanoporetech.com) by email or use LiveChat in the Nanopore Community.

Issues during DNA/RNA extraction and library preparation for Kit 14:
Low sample quality 
        
  Observation 
  Possible
  cause 
  Comments and
  actions
  
  Low DNA purity (Nanodrop reading
    for DNA OD 260/280 is <1.8 and OD
    260/230 is <2.0–2.2)
  The
  DNA extraction
    method does not
    provide the required
    purity
  The
  effects of contaminants are shown in theContaminants
    document. Please try an alternative extraction method that
    does not result in contaminant carryover.
    Consider performing an additional SPRI clean-up step.
  
  Low RNA integrity (RNA integrity
    number <9.5 RIN, or the rRNA band
    is shown as a smear on the gel)
  The
  RNA degraded
    during extraction
  Try
  a different RNA extraction method. For more info on RIN,
    please see the RNA Integrity Number document. Further
    information can be found in the DNA/RNA Handling
  page.
  
  RNA has a shorter than expected
    fragment length
  The
  RNA degraded
    during extraction
  Try
  a different RNA extraction method. For more info on RIN,
    please see the RNA Integrity Number document. Further
    information can be found in the DNA/RNA Handling
  page.
    We recommend working in an RNase-free environment, and to
    keep your lab equipment RNase-free when working with RNA.
  
 
Low DNA recovery after AMPure bead clean-up
Observation Possible cause Comments and actionsLowrecoveryDNA loss due to alower than intendedAMPure beads-tosample ratio1. AMPure beads settle quickly, so ensure they are well resuspended before adding them tothe sample.2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any sizewill be lost during the clean-up.LowrecoveryDNA fragments areshorter thanexpectedThe lower the AMPure beads-to-sample ratio, the more stringent the selection against shortfragments. Please always determine the input DNA length on an agarose gel (or other gelelectrophoresis methods) and then calculate the appropriate amount of AMPure beads touse.Lowrecoveryafter endprepThe wash step usedethanol <70%DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correctpercentage
  
 
Issues during the sequencing run for Kit 14:
Fewer pores at the start of sequencing than after Flow Cell Check 
Observation PossiblecauseComments and actionsMinKNOWreported alower numberof pores at thestart ofsequencingthan thenumberreported bythe Flow CellCheckAn air bubblewas introducedinto thenanopore arrayAfter the Flow Cell Check it is essential to remove any air bubbles near the priming port beforepriming the flow cell. If not removed, the air bubble can travel to the nanopore array andirreversibly damage the nanopores that have been exposed to air. The best practice toprevent this from happening is demonstrated in this video.MinKNOWreported alower numberof pores at thestart ofsequencingthan thenumberreported bythe Flow CellCheckThe flow cell isnot correctlyinserted intothe deviceStop the sequencing run, remove the flow cell from the sequencing device and insert it again,checking that the flow cell is firmly seated in the device and that it has reached the targettemperature. If applicable, try a different position on the device (GridION/PromethION).MinKNOWreported alower numberof pores at thestart ofsequencingthan thenumberreported bythe Flow CellCheckContaminationsin the librarydamaged orblocked theporesThe pore count during the Flow Cell Check is performed using the QC DNA molecules presentin the flow cell storage buffer. At the start of sequencing, the library itself is used to estimatethe number of active pores. Because of this, variability of about 10% in the number of pores isexpected. A significantly lower pore count reported at the start of sequencing can be due tocontaminants in the library that have damaged the membranes or blocked the pores.Alternative DNA/RNA extraction or purification methods may be needed to improve the purityof the input material. The effects of contaminants are shown in the Contaminants Know-howpiece. Please try an alternativeextraction method that does not result in contaminantcarryover.
MinKNOW script failed
Observation PossiblecauseComments and actionsMinKNOWshows"Scriptfailed"Restart the computer and then restart MinKNOW. If the issue persists, please collect theMinKNOW logfiles and contact Technical Support. If you do not have another sequencing device available, werecommend storing the flow cell and the loaded library at 4°C and contact Technical Support for furtherstorage guidance.
Pore occupancy below 40%
Observation Possible cause Comments and actionsPoreoccupancy<40%Not enough library was loaded on theflow cell10–20 fmol of good quality library can be loaded on to a MinION/GridIONflow cell. Please quantify the library before loading and calculate molsusing tools like the Promega Biomath Calculator, choosing "dsDNA: µgto pmol"
 Shorter than expected read length
        
  Observation 
  Possible
  cause 
  Comments and
  actions
  
  Shorter than
    expected read length
  Unwanted
  fragmentation
    of DNA sample
  Read
  length reflects input DNA fragment length. Input DNA can be
    fragmented during extraction and library prep.
    1. Please review the Extraction Methods in the Nanopore Community for best practice for
  extraction.
    2. Visualise the input DNA fragment length distribution on an agarose
  gel
    before proceeding to the library prep.
    In the image above, Sample 1 is of high molecular weight, whereas Sample 2
  has been fragmented.
    3. During library prep, avoid pipetting and vortexing when mixing
  reagents.
    Flicking or inverting the tube is sufficient.
  
 
 
Large proportion of unavailable pores 
Observation PossiblecauseComments and actionsLarge proportion of unavailable pores (shown asblue in the channels panel and pore activity plot)The pore activity plot above shows an increasingproportion of "unavailable" pores over time.Contaminantsare present inthe sampleSome contaminants can be cleared from the pores bythe unblocking function built into MinKNOW. If this issuccessful, the pore status will change to "sequencingpore". If the portion of unavailable pores stays large orincreases:1. A nuclease flush using the Flow Cell Wash Kit (EXPWSH004) can be performed, or2. Run several cycles of PCR to try and dilute anycontaminants that may be causing problems.
Large proportion of inactive pores
Observation PossiblecauseComments and actionsLarge proportion of inactive/unavailable pores(shown as light blue in the channels panel andpore activity plot. Pores or membranes areirreversibly damaged)Air bubbleshave beenintroducedinto the flowcellAir bubbles introduced through flow cell priming andlibrary loading can irreversibly damage the pores.Watch the Priming and loading your flow cellvideo forbest practiceLarge proportion of inactive/unavailable pores Certaincompoundsco-purifiedwith DNAKnown compounds, include polysaccharides, typicallyassociate with plant genomic DNA.1. Please refer to thePlant leaf DNA extractionmethod.2. Clean-up using the QIAGEN PowerClean Pro kit.3. Perform a whole genome amplification with theoriginal gDNA sample using the QIAGEN REPLI-g kit.Large proportion of inactive/unavailable pores Contaminantsare present inthe sampleThe effects of contaminants are shown in theContaminants Know-how piece. Please try analternative extraction method that does not result incontaminant carryover.
Reduction in sequencing speed and q-score later into the run
 

Lower than expected sequencing depth in adaptive sampling
 

Disproportionate sequencing of individual barcoded samples within a pool
 

Temperature fluctuation
 

Failed to reach target temperature
 



[STEPS]
SECTION: Prepare for your experiment
1. This protocol is based on ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-24-NBE_9169_v114_revF_15Sep2022-promethion with some modifications for adaptive sampling. 
  

Refer to the ONT notes on adaptive sampling for further information and creating a .BED file.
  
  
SECTION: Prepare samples for your experiment
3. This protocol is for barcoding 4-5 samples per flow cell.
Only 2 flow cells may be run simultaneously with high accuracy basecalling required for adaptive sampling. 
Extract DNA, and check its length, quantity and purity. 
  
  
SECTION: Prepare samples for your experiment
5. Perform a 1X ProNex‱ bead clean-up on fragmented gDNA

SECTION: DNA repair and end-prep
7. Thaw the AMPure XP Beads (AXP) at Room temperature 
Thaw NEBNext FFPE DNA Repair Buffer, Ultra II End-Prep Reaction Buffer, Ultra II End-Prep Enzyme Mix and NEBNext FFPE DNA Repair Mix on ice.

Flick and/or invert the reagent tubes to ensure they are well mixed. Spin down tubes before opening.

 


 

SECTION: DNA repair and end-prep
8. Optional addition of DNA Control Sample (DCS).

 

SECTION: DNA repair and end-prep
9. Prepare a mastermix of the end-prep and DNA repair reagents for the total number of samples and add 3 µL to each sample tube. 

      
  Reagent 
  Volume
  
  DNA sample 
  11
  µl
  
  Diluted DNA Control Sample (DCS)
  1
  µl
  
  NEBNext FFPE DNA Repair Buffer 
  0.875
  µl
  
  Ultra II End-prep Reaction Buffer 
  0.875
  µl
  
  Ultra II End-prep Enzyme Mix 
  0.75
  µl
  
  NEBNext FFPE DNA Repair Mix 
  0.5
  µl
  
  Total 
  15
  µl
  
 
 

Pipette mix 10 - 20 times and spin down in a centrifuge.
SECTION: DNA repair and end-prep
10. Using a thermal cycler, incubate at 20 °C for 30 min and 65 °C for 5 min.

SECTION: DNA repair and end-prep
11. Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: DNA repair and end-prep
12. Resuspend the AMPure XP beads (AXP) by vortexing.
SECTION: DNA repair and end-prep
13. Add 15 µL of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.

SECTION: DNA repair and end-prep
14. Incubate on a Hula rotator mixer for 10 min at Room temperature.

SECTION: DNA repair and end-prep
15. Prepare sufficient fresh 80% ethanol in nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
SECTION: DNA repair and end-prep
16. Spin down the samples and pellet the beads on a magnet for 2 min until the eluate is clear and colourless. Keep the tubes on the magnet, pipette off the supernatant and retain (SN2)

SECTION: DNA repair and end-prep
17. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

 

SECTION: DNA repair and end-prep
18. Repeat the previous step   

SECTION: DNA repair and end-prep
19. Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Do not allow the pellet to dry out.

SECTION: DNA repair and end-prep
20. Remove the tubes from the magnetic rack and resuspend the pellet in 10 µL nuclease-free water. Spin down and incubate for 10 min at 37 °C.

SECTION: DNA repair and end-prep
21. Pellet the beads on a magnet for 2 min until the eluate is clear and colourless.
SECTION: DNA repair and end-prep
22. Remove and retain 10 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: DNA repair and end-prep
23. Quantify 1 µL of each eluted sample using a Qubit fluorometer. 

 

SECTION: Native barcode ligation
25. Thaw the NEB Blunt/TA Ligase Master Mix at Room temperature, spin briefly and mix by performing 10 full volume pipette mixes. Store on ice.
Thaw the EDTA at Room temperature and mix by vortexing. Then spin down and place on ice.
Thaw the required number of Native Barcodes (NB01-24) at Room temperature. Individually mix the barcodes by pipetting, spin down and place them on ice.

 

SECTION: Native barcode ligation
26. In clean 0.2 ml PCR-tubes add the reagents in the following order:
Between each addition, pipette mix 10 - 20 times 
      
  Reagent 
  Volume
  
  End-prepped DNA 
  7.5
  µl
  
  Native Barcode (NB01-24) 
  2.5
  µl
  
  Blunt/TA Ligase Master
    Mix
  10
  µl
  
  Total 
  20
  µl
  
 
SECTION: Native barcode ligation
27. Thoroughly mix the reaction by gently pipetting and briefly spinning down.
SECTION: Native barcode ligation
28. Incubate for 20 min at Room temperature.

SECTION: Native barcode ligation
29. Add 2 µL EDTA to each reaction and mix thoroughly by pipetting and spin down briefly.
 
 

SECTION: Native barcode ligation
30. Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube. 
            
  
  Volume per sample 
  For 6
  samples 
  For 12
  samples 
  For 24 samples
  
  Total
  volume for preps usingclear cap EDTA
  22
  µl 
  132
  µl 
  264
  µl 
  528
  µl
  
  Total volume for preps usingblue cap
  EDTA 
  24
  µl 
  144
  µl 
  288
  µl 
  576
  µl
  
 
 

SECTION: Native barcode ligation
31. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Native barcode ligation
32. Add 0.8X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.
 

SECTION: Native barcode ligation
33. Incubate on a Hula rotator mixer for 10 min at Room temperature.

SECTION: Native barcode ligation
34. Prepare 2 mL of fresh 80% ethanol in nuclease-free water.

SECTION: Native barcode ligation
35. Spin down the sample and pellet on a magnet for 5 min. Keep the tube on the magnetic rack until the eluate is clear and colourless. Pipette off the supernatant and retain (SN3).

SECTION: Native barcode ligation
36. Keep the tube on the magnetic rack and wash the beads with 700 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

 

SECTION: Native barcode ligation
37. Repeat the previous step   

SECTION: Native barcode ligation
38. Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Do not dry the pellet to dry out.

SECTION: Native barcode ligation
39. Remove the tube from the magnetic rack and resuspend the pellet in 32 µL nuclease-free water by gently flicking.

SECTION: Native barcode ligation
40. Incubate for 10 min at 37 °C. 
Every 2 minutes, agitate the sample by gently flicking for 10 s to encourage DNA elution.

SECTION: Native barcode ligation
41. Pellet the beads on a magnetic rack for 2 min until the eluate is clear and colourless.

SECTION: Native barcode ligation
42. Remove and retain 32 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Native barcode ligation
43. Quantify 1 µL of eluted sample using a Qubit fluorometer.

 

 

SECTION: Adapter ligation and clean-up
44. Thaw NEBNext Quick Ligation Reaction Buffer (5X) at Room temperature. Spin down and ensure the reagent is fully mixed by performing 10 full volume pipette mixes. Store on ice.

 

Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.

 

Thaw the Long Fragment Buffer (LFB) and Elution Buffer (EB) at Room temperature and mix by vortexing. Then spin down and place on ice.
 

SECTION: Adapter ligation and clean-up
45. In a 1.5 ml Eppendorf LoBind tube, mix in the following order:
Between each addition, pipette mix 10 - 20 times. 
      
  Reagent 
  Volume
  
  Pooled barcoded sample 
  30
  µl
  
  Native Adapter (NA) 
  5
  µl
  
  NEBNext Quick Ligation Reaction Buffer (5X)
  10
  µl
  
  Quick T4 DNA Ligase 
  5
  µl
  
  Total 
  50
  µl
  
 Thoroughly pipette mix the reaction and briefly spin down.
SECTION: Adapter ligation and clean-up
46. Incubate the reaction for 20 min at Room temperature.


SECTION: Adapter ligation and clean-up
47. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Adapter ligation and clean-up
48. Add 40 µL of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.

49. Incubate on a Hula rotator mixer for 10 min at Room temperature.

SECTION: Priming and loading the PromethION Flow Cell
59. Remove flow cell/s from the fridge. Wait 20 min before inserting into the PromethION to allow the flow cell to come to Room temperature.

 

 

Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT) and one tube of Flow Cell Flush (FCF) at Room temperature, mix by vortexing, spin down and store on ice.

SECTION: Priming and loading the PromethION Flow Cell
60. To prepare the flow cell priming mix, combine Flow Cell Tether (FCT) and Flow Cell Flush (FCF), as directed below:
SECTION: Priming and loading the PromethION Flow Cell
60.1. Single-use tubes format:

Add 30 µL Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).

SECTION: Priming and loading the PromethION Flow Cell
60.2. Sequencing Auxiliary Vials V14 (EXP-AUX003) format:

In a clean suitable tube for the number of flow cells, combine and mix the following reagents:
      
  Reagent 
  Volume per flow
  cell
  
  Flow Cell Flush (FCF) 
  1,170
  µl
  
  Flow Cell Tether (FCT)
  30
  µl
  
  Total volume 
  1,200
  µl
  

SECTION: Priming and loading the PromethION Flow Cell
61. Load the flow cell(s) into the docking ports on the PromethION 24/48.
SECTION: Priming and loading the PromethION Flow Cell
61.1. Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
SECTION: Priming and loading the PromethION Flow Cell
61.2. Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
 
 

 

SECTION: Priming and loading the PromethION Flow Cell
62. Turn the valve clockwise to expose the inlet port.
 
SECTION: Priming and loading the PromethION Flow Cell
63. After opening the inlet port, draw back a small volume to remove any air bubbles:
SECTION: Priming and loading the PromethION Flow Cell
63.1. Set a P1000 pipette tip to 200 µL.

SECTION: Priming and loading the PromethION Flow Cell
63.2. Insert the tip into the inlet port.
SECTION: Priming and loading the PromethION Flow Cell
63.3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.
 
 

SECTION: Priming and loading the PromethION Flow Cell
64. Load 500 µL of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait 5 min. 
During this time, prepare the library for loading  
 

SECTION: Priming and loading the PromethION Flow Cell
65. 
 

In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows: 
      
  Reagent 
  Volume per flow
  cell
  
  Sequencing Buffer (SB) 
  100
  µl
  
  Library Beads (LIB) thoroughly mixed before use, or Library
  Solution (LIS)
  68
  µl
  
  DNA library 
  32
  µl
  
  Total 
  200
  µl
  
 
 

SECTION: Priming and loading the PromethION Flow Cell
66. Complete the flow cell priming by slowly loading 500 µL of the priming mix into the inlet port.
 
Wait 5 min  before loading library

SECTION: Priming and loading the PromethION Flow Cell
67. Mix the prepared library gently by pipetting up and down just prior to loading.
Using a P1000, insert the pipette tip into the inlet port and load 200 µL of library.
 
SECTION: Priming and loading the PromethION Flow Cell
68. Close the valve to seal the inlet port. 

 
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71. Select 'Start Sequencing' on the start page of the MinKNOW software to set up the sequencing parameters for the experiment:
SECTION: Ending the experiment
83. Flow cells are unlikely to be re-useable given the nature of adaptive sampling. However, if you would like to reuse the flow cell, follow the Flow Cell Wash steps   . Store the washed flow cell at 2 °C-8 °C.
SECTION: Prepare samples for your experiment
4. High molecular weight DNA may require shearing prior to adaptive sampling. If shearing is not required, proceed with clean-up and volume reduction   
Shear 6 µg  gDNA using Diagenode Megaruptor‱ 3 DNA shearing kit (and DNAFluid+ kit for viscous samples) to obtain uniform fragment lengths of 12-15 kb. 
Check fragment length using Agilent Femto Pulse Genomic 165 kb kit (or similar electrophoresis method). 
  
 

SECTION: Prepare for your experiment
2. Ensure you have your Native Barcoding Kit 24 V14 (SQK-NBD114.24), the correct equipment and third-party reagents.

Check your PromethION R10.4.1 flow cell/s (FLO-PRO114M) to ensure there are >5000 pores.
SECTION: Prepare samples for your experiment
5.1. Allow ProNex‱ beads to get to Room temperature and resuspend by vortexing.
SECTION: Prepare samples for your experiment
5.2. Add 1X the volume of resuspended ProNex‱ beads to gDNA and mix by flicking the tube.
SECTION: Prepare samples for your experiment
5.3. Incubate on a Hula rotator mixer for 10 min at Room temperature.
SECTION: Prepare samples for your experiment
5.4. Meanwhile, prepare sufficient fresh 80% ethanol in nuclease-free water to completely cover the samples. Allow enough for two washes, with some excess.
SECTION: Prepare samples for your experiment
5.5. Spin down the samples and pellet the beads on a magnet for 2 min until the eluate is clear and colourless. Keep the tubes on the magnet, pipette off the supernatant and retain (SN1).

 

SECTION: Prepare samples for your experiment
5.6. Keep the tube on the magnet and wash the beads with enough freshly prepared 80% ethanol to cover the pellet. Remove the ethanol using a pipette and discard.

 

SECTION: Prepare samples for your experiment
5.7. Repeat the previous step   

SECTION: Prepare samples for your experiment
5.8. Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off and discard any residual ethanol. Do not allow the pellet to dry out.
SECTION: Prepare samples for your experiment
5.9. Remove the tubes from the magnetic rack and resuspend the pellet in 25 µL nuclease-free water. Spin down and incubate for 10 min at Room temperature.

 

SECTION: Prepare samples for your experiment
5.10. Pellet the beads on a magnet for 2 min until the eluate is clear and colourless.
SECTION: Prepare samples for your experiment
5.11. Remove and retain 25 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Prepare samples for your experiment
5.12. Quantify 1 µL of each eluted sample using a Qubit fluorometer. 

 

SECTION: DNA repair and end-prep
24. Take forward an equimolar mass of each sample to be barcoded into the native barcode ligation step:
Use an online weight to molar quantity calculator to identify which sample has the lowest molar concentration in 7.5 µL. Dilute other samples to the same molar quantity in 7.5 µL.

 

 

SECTION: Ending the experiment
84. Alternatively, flush the flow cell/s with deionised water and store ready to send back to Oxford Nanopore. 

SECTION: Priming and loading the PromethION Flow Cell
70. Wait a minimum of 30 min after loading the flow cells with library before initiating sequencing to increase the sequencing output.

50. Spin down the samples and pellet the beads on a magnet for 2 min until the eluate is clear and colourless. Keep the tubes on the magnet, pipette off the supernatant and retain (SN4)

51. Wash the beads by adding 125 µL Long Fragment Buffer (LFB). 
Remove from the magnet, flick the beads to resuspend, spin down, incubate for 2 min at Room temperature before returning the tube to the magnetic rack. 
Pellet the beads on a magnetic rack for 2 min until the eluate is clear and colourless. Remove the supernatant using a pipette and discard.
 

52. Repeat the previous step   

53. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Do not allow  the pellet to dry out.
54. Remove the tube from the magnetic rack and resuspend the pellet in 25 µL Elution Buffer (EB). Spin down and incubate for 10 min at 37 °C. Every 2 minutes, agitate the sample by gently flicking for 10 s to encourage DNA elution.
55. Pellet the beads on a magnet for 2 min until the eluate is clear and colourless.

56. Remove and retain 25 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
57. Quantify 1 µL of eluted sample using a Qubit fluorometer.
 

58. Prepare the final library to 50 fmol in 32 µL of Elution Buffer. 
 

Store the library on ice or at 4°C until ready to load.
 

SECTION: Prepare samples for your experiment
6. If necessary, dilute gDNA with nuclease free water to obtain 2X 12 µL aliquots per sample at a concentration of 1200-1500 ng in clean 0.2 ml thin-walled PCR tubes.

 

SECTION: Priming and loading the PromethION Flow Cell
69. Install the light shield on your flow cell as follows: 

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.
 
 
 

Close the PromethION lid when ready to start a sequencing run on MinKNOW.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
72. Click on the flow cell to check the number of active pores. 

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.1. Type in the experiment name appended with a run number for the chosen flow cell/s. Label sample ID's.
 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.2. Select the Native Barcoding Kit 24 V14 (SQK-NBD114.24) used to prepare the library.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.3. Set up the run options:
run limit: 96 hours
Minimum read length: 200 bp
Adaptive sampling: select 'Enrich or deplete sequences'. 
Upload an alignment reference (e.g. hg38 FASTA file) and specific sequence coordinates of interest (e.g. SCA4_human_hg38.bed). 
Select 'enrich alignment matches'.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.4. Turn 'barcode balancing' off.

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.5. 'Active channel selection' is on by default. 

 

The time between pore scans is 1.5 hours by default.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.6. Turn 'Reserved pore' option off to front-load sequence data acquisition.

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.7. 'Basecalling' is on by default. Click "Edit options" to specify the basecall model as 'High accuracy Basecalling'.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.8. 'Modified bases' is off by default. 

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.9. 'Barcoding' is on by default.

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.10. 'Alignment' is optional.

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.11. Select 'output location'.

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.12. Select 'output format':
Raw reads default is .POD5
Basecalled reads default is .FASTQ
Aligned reads (if selected) default is .BAM 

 

SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.13. 'Filtering' is at Q9 by default for the high accuracy basecalling.
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
71.14. Click 'Start' to review all run options selected. 
Edits can be made by selecting the 'Edit' button.
Select 'Advanced run options' to view the extra options selected.
Click 'Start' to begin sequencing
SECTION: Data acquisition and high accuracy basecalling for adaptive sampling
73. Monitor translocation speed, quality score and data output during the sequencing run. 
If translocation speed ≤350 bps, quality score begins to drop toward Q9 and there is little increase in data output: 

pause sequencing
Perform a flow cell wash
Prime flow cell
load 50 fmol prepared library
restart sequencing
SECTION: Performing a flow cell wash
74.   
Place the tube of Wash Mix (WMX) on ice. Do not vortex.
Thaw one tube of Wash Diluent (DIL) at room temperature. Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.
SECTION: Performing a flow cell wash
75. In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:

 
Mix well by pipetting and place on ice. Do not vortex.
SECTION: Performing a flow cell wash
76. Pause the sequencing run.
SECTION: Performing a flow cell wash
77. Ensure the inlet port is closed and remove any waste fluid from the waste port labelled 2 on the flow cell.
SECTION: Performing a flow cell wash
78. Open the inlet port and draw back a small volume to remove any air bubbles
  

SECTION: Performing a flow cell wash
79. Load 400 µL of the prepared Flow Cell Wash Mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait 60 min. 
SECTION: Performing a flow cell wash
80. Prepare Priming Mix and library, load flow cell. 
  

 

SECTION: Performing a flow cell wash
81. Restart sequencing. 

 

SECTION: Performing a flow cell wash
82. Monitor translocation speed, quality score and data output during the sequencing run. 
If translocation speed ≤350 bps, quality score begins to drop toward Q9 and there is little increase in data output perform a flow cell wash, re-prime the flow cell, load 50 fmol prepared library and restart sequencing  
SECTION: Data Analysis
85. Data analysis pipeline is available through https://github.com/egustavsson/long-read_SV_calling.git