TITLE: ssUMI: high-throughput long-read sequencing workflow for highly-accurate near full-length 16S rRNA genes on the ONT platform
AUTHORS: Xuan Lin, Katherine Waring, John Tyson, Ryan Ziels

[DESCRIPTION]
This is the online protocol for near full-length 16S rRNA amplicon sequencing with unique molecule identifiers (ssUMI) on the Nanopore platform. Sequencing libraries are prepared with Oxford Nanopore Native Barcoding kit 96 V12 or V14 (SQK-NBD112.96 or SQK-NBD114.96) for sequencing on PromethION platforms in super high accuracy mode (260 bps).

The near full-length 16S rRNA amplicons are generated with primers 8F: AGRGTTYGATYMTGGCTCAG and 1391R: GACGGGCGGTGWGTRCA; the UMI design was adapted from (Karst and Ziels, 2021) and the Oxford Nanopore community protocol. 

For more details, please check our preprint: https://www.biorxiv.org/content/10.1101/2023.06.19.544637v1

[BEFORE_START]
This protocol was developed with 96 samples, and we highly recomand using multi-channel pipettes and/or an automatic liquid handler.

Time can be saved by preparing master mixes first, before PCR steps. The master mix for PCR steps should be prepared in a Master Mix (PCR) Hood. To avoid cross-contamination make sure that your original stock reagents have no contact with any amplified DNA material.


A Negative Control (nuclease-free H2O) should be included.
Keep the enzymes on ice and thaw the other reagents at room temperature, mix, then spin down before placing on ice. 


All steps after ssUMI-EarlyPCR2 should be performed in post-PCR area.

[STEPS]
SECTION: Sample pre-dilution and ddPCR quantification of starting material
1. Estimate the full-length 16S rRNA concentration in the DNA extract (e.g., sample), and dilute the sample to 20000 copies/μL with  .


 


SECTION: ssUMI-PCR1: UMI tagging and cleanup
3. In this step, the near full-length 16S rRNA gene primers containing UMIs are annealed to both ends of the DNA template using 2 rounds of PCR. Only one copy of a dual-end UMI-tagged amplicon will be generated for each input molecule.
SECTION: ssUMI-EarlyPCR2: amplification of UMI-tagged amplicons
5. In this step, the dual-UMI-tagged near full-length 16S rRNA gene amplicons are further amplified with PCR.
SECTION: ssUMI-LatePCR2: amplification of UMI-tagged amplicons
7. Similar to ssUMI-EarlyPCR2, the dual-UMI-tagged near full-length 16S rRNA gene amplicons are further amplified with 15 cycles of PCR.
6. ssUMI-EarlyPCR2 cleanup using    with a bead-to-sample ratio of 0.6.

 

8. ssUMI-LatePCR2 cleanup using   with a bead-to-sample ratio of 0.6.

 

SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10. DNA End-prep with   

Reagent preparation before starting this step
1. Thaw the NEBNext Ultra II End Prep Reaction Buffer on ice, check for any visible precipitate; vortexing for at least 30 seconds may be required to solubilise all precipitate.
2. Place the Ultra II End Prep Enzyme Mix on ice 

 

11. Native barcode ligation with Native Barcoding Kit 12 or Kit 14.

Reagent preparation before starting this step
1. Thaw   at Room temperature, spin down and mix by performing 10 full volume pipette mixes, then place on ice.

2. Thaw the AMPure XP Beads (AXP) at Room temperature and mix by vortexing. Keep the beads at Room temperature.

3. Thaw the EDTA (provided with the sequencing kit) at Room temperature and mix by vortexing. Then spin down and place on ice .

4. Thaw the Native Barcodes NB01-96 at Room temperature. Individually mix the barcodes by pipetting, spin down, and place them on ice.

12. Native Barcoding reaction cleanup with 0.4x AMPure XP Beads.

Before start of this step:
1. Prepare 2 mL fresh 80% ethanol in nuclease-free water.
2. Pre-heat an incubator to 37 °C.

SECTION: Sample pre-dilution and ddPCR quantification of starting material
2. Run ddPCR quantification with 2.5 µL  diluted DNA sample. 

SECTION: Sample pre-dilution and ddPCR quantification of starting material
2.1. Prepare the ddPCR mastermix in a PCR hood, vortex to mix and dispense 19.5 µL into each well of the   . Each component is thawed at Room temperature , vortexed to mix, then spun down. A 10% pipetting loss should be included when preparing mastermix for multiple samples.

 

SECTION: Sample pre-dilution and ddPCR quantification of starting material
2.2. Add 2.5 µL to each well, seal the plate with   , vortex to mix, and spin down.

SECTION: Sample pre-dilution and ddPCR quantification of starting material
2.3. Load the plates on the QX200 AutoDG Droplet Digital PCR System, and run droplet generation.
SECTION: Sample pre-dilution and ddPCR quantification of starting material
2.4. Run PCR in a deep-well thermalcycler using the following program (set total volume as 40 µL) :

 

 

SECTION: Sample pre-dilution and ddPCR quantification of starting material
2.5. Count droplets in QX200 AutoDG Droplet Digital PCR System using manufacturer's protocols.
SECTION: ssUMI-PCR1: UMI tagging and cleanup
3.1. Adjust the input DNA concentration to 5000 copies/μL with nuclease-free water (100,000 16S rRNA gene copies in 20 µL) based on the ddPCR measured near full-length 16S rRNA gene copy numbers (see Step 2.5).
SECTION: ssUMI-PCR1: UMI tagging and cleanup
3.2. Prepare ssUMI-PCR1 mastermix in a PCR hood, flick the tube to mix and dispense 30 µL into each well of a 96-well PCR plate. Each component is thawed on ice , mixed by flicking the tube, then spun down. A 10% pipetting loss should be included when preparing mastermix for multiple samples.

 

SECTION: ssUMI-PCR1: UMI tagging and cleanup
3.4. Run PCR in a thermocycler using the following program (total volume: 50 µL) :

 

9. Quantify 1 µL  of the cleaned ssUMI-LatePCR2 product using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. Amplicons should have concentration higher than 4 ng/μL 

 



6.1. Homogenize   solution by vortexing.
4.14. Pipette off the 18 µL supernatant to a new 96-well PCR plate.

4.13. Place the plate on the magnetic rack to pellet the beads and wait until the eluate is clear and colourless.
4.12. Incubate at Room temperature for 5 min .

4.11. Remove the plate from the magnetic rack and elute the purified DNA by adding 20 µL  and mix by pipetting.

4.10. Let the beads air dry for a maximum of 2 min (but do not overdry the beads to the point of cracking).
4.9. Spin down and place the plate back on the magnetic rack, then remove residual ethanol with a smaller pipette.


 

4.8. Repeat the washing steps (Steps 4.5-4.7).

4.7. Wait 30 seconds and discard the ethanol.
4.6. Wash beads by adding 200 µL along the opposite side of the beads.

4.5. Keep the plate on the magnetic rack, carefully discard the supernatant.
4.4. Place the plate on a magnetic rack to pellet the beads, and wait until the solution is clear on the side of each well (~5 min).
4.3. Spin down, and then incubate at Room temperature for 10 min.

4.2. Add 30 µL to the 50 µL (ratio 0.6) and mix by pipetting or brief vortexing.

4.1. Homogenize   solution by vortexing.

4. ssUMI-PCR1 cleanup using    with a bead-to-sample ratio of 0.6.

6.2. Add 30 µL to the 50 µL (ratio 0.6) and mix by pipetting or brief vortexing.
6.3. Spin down, and then incubate at Room temperature for 10 min.

6.4. Place the plate on a magnetic rack to pellet the beads, and wait until the solution is clear on the side of each well (~5 min).
6.5. Keep the plate on the magnetic rack, carefully discard the supernatant.
6.6. Wash beads by adding 200 µL along the opposite side of the beads.

6.7. Wait 30 seconds and discard the ethanol.
6.8. Repeat the washing steps (Steps 6.5-6.7).
6.9. Spin down and place the plate back on the magnetic rack, then remove residual ethanol with a smaller pipette.
6.10. Let the beads air dry for a maximum of 2 min (but do not overdry the beads to the point of cracking).
6.11. Remove the plate from the magnetic rack and elute the purified DNA by adding 20 µL  and mix by pipetting.
6.12. Incubate at Room temperature for 5 min .
6.13. Place the plate on the magnetic rack to pellet the beads and wait until the eluate is clear and colourless.
6.14. Pipette off the 18 µL supernatant to a new 96-well PCR plate.


 

8.1. Homogenize   solution by vortexing.
8.2. Add 30 µL to the 50 µL (ratio 0.6) and mix by pipetting or brief vortexing.
8.3. Spin down , and then incubate at Room temperature for 10 min.

8.4. Place the plate on a magnetic rack to pellet the beads, and wait until the solution is clear on the side of each well (~5 min).
8.5. Keep the plate on the magnetic rack, carefully discard the supernatant.
8.6. Wash beads by adding 200 µL along the opposite side of the beads.

8.7. Wait 30 seconds and discard the ethanol.
8.8. Repeat the washing steps (Steps 8.5-8.7).
8.9. Spin down and place the plate back on the magnetic rack, then remove residual ethanol with a smaller pipette.
8.10. Let the beads air dry for a maximum of 2 min (but do not overdry the beads to the point of cracking).
8.11. Remove the plate from the magnetic rack and elute the purified DNA by adding 20 µL  and mix by pipetting.
8.12. Incubate at Room temperature for 5 min .
8.13. Place the plate on the magnetic rack to pellet the beads and wait until the eluate is clear and colourless.
8.14. Pipette off the 18 µL supernatant to a new 96-well PCR plate.

SECTION: ssUMI-EarlyPCR2: amplification of UMI-tagged amplicons
5.1. Prepare ssUMI-PCR2 mastermix in PCR hood, flick the tube to mix. Each component is thawed on ice , mixed by flicking the tube, then spun down. A 10% pipetting loss should be included when preparing mastermix for multiple samples.


 

SECTION: ssUMI-EarlyPCR2: amplification of UMI-tagged amplicons
5.3. Spin down and run PCR in a thermocycler using the following program (total volume: 50 µL) :

 


SECTION: ssUMI-PCR1: UMI tagging and cleanup
3.3. Add 20 µL (100,000 full-length 16S rRNA gene copies) into each well of a 96-well PCR plate, 

SECTION: ssUMI-LatePCR2: amplification of UMI-tagged amplicons
7.1. Prepare the same mastermix as in Step 5.1 (ssUMI-PCR2 mastermix) in PCR hood, flick the tube to mix. Each component is thawed on ice , mixed by flicking the tube, then spun down. A 10% pipetting loss should be included when preparing mastermix for multiple samples.

 

SECTION: ssUMI-LatePCR2: amplification of UMI-tagged amplicons
7.3. Spin down and run PCR in a thermocycler using the following program (total volume: 50 µL) :

 

SECTION: ssUMI-LatePCR2: amplification of UMI-tagged amplicons
7.2. Dispense 32 µL into each well of the 96-well PCR plate containing 18 μL cleaned ssUMI-EarlyPCR2 product.

 

SECTION: ssUMI-EarlyPCR2: amplification of UMI-tagged amplicons
5.2. Dispense 32 µL into each well of the 96-well PCR plate containing 18 μL cleaned ssUMI-PCR1 product.
SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10.1. In a clean 96-well plate, aliquot 50 fmol(46.4 ng) of amplicon per sample (based on quantification in Step 9).

SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10.2. Make up each sample to 12.5 µL using nuclease-free water. Mix gently by pipetting and spin down.

SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10.3. Prepare End-prep mastermix, mix by pipetting 10-20 times. A 10-15% pipetting loss should be included when preparing mastermix for multiple samples.

 

SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10.4. Add 2.5 µL to each well containing DNA amplicons, pipetting 10-20 times to mix and spin down briefly.

SECTION: Nanopore sequencing library preparation using Native Barcoding Kit 12/14
10.5. Using a thermocycler, incubate at 20 °C for 5 min and 65 °C for 5 min.


 

11.1. In a new 96-well plate, add the reagents in the following order per well (select unique barcode for each sample on the plate). Between each addition, pipette mix 10 times.

 

11.2. Thoroughly mix the reaction by gently pipetting 10 times and then briefly spin down.
11.3. Incubate for 20 min at Room temperature.

11.4. Add 1 µL to each well and mix thoroughly by pipetting and spin down briefly.

11.5. Pool all barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
12.1. Resuspend the AMPure XP Beads (AXP) by vortexing.

12.2. Add 422 µL (for 96 samples) AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.4x clean.

12.3. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature.

12.4. Spin down the sample and pellet on a magnet for 5 min. Keep the plate on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.

12.5. Keep the tube on the magnetic rack and wash the beads with 700 µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

 

12.6. Repeat the previous step (step 12.5).

12.7. Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not overdry the pellet to the point of cracking.
12.8. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL 35 μl nuclease-free water by gently flicking or pipetting.

12.9. Incubate for 10 min at 37 °C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.

12.10. Pellet the beads on a magnetic rack until the eluate is clear and colourless.
12.11. Remove and retain 35 µLof eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

13. Quantify 1 µL  of the eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. 

 

14. Nanopore sequencing adapter ligation

Reagent preparation before starting this step
1. Thaw the   at Room temperature , spin down and mixed by performing 10 full volume pipette mixes.

2. Spin down the Adapter Mix II (AMII H, Kit 12) or Native Adapter (NA, Kit 14) and  , pipette mix and place on ice.

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

 

14.1. In a 1.5 ml Eppendorf LoBind tube, mix in the following order. Between each addition, pipette mix 10 times.

 

14.2. Thoroughly mix the reaction by gently pipetting and briefly spinning down.
14.3. Incubate the reaction for 20 min at Room temperature.

15. Adapter Ligation reaction cleanup with 0.4x AMPure XP Beads.

Before start of this step:
Pre-heat an incubator to 37 °C.

15.1. Resuspend the AMPure XP Beads (AXP) by vortexing.

15.2. Add 20 µLof resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.

15.3. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature.

15.4. Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
15.5. Wash the beads by adding 125 µL Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

15.6. Repeat the previous step (Step 15.5).
15.7. Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
15.8. Remove the tube from the magnetic rack and resuspend pellet in 25 µL Elution Buffer (EB).

15.9. Spin down and incubate for 10 min at 37 °C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.

15.10. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
15.11. Remove and retain 25 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

16. Quantify 1 µL of the eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. Then make up the library to 32 μl at 10-20 fmol (18.4 ng).