TITLE: Sequencing Bacterial Isolates from Contaminated Food Samples with ONT
AUTHORS: Sarah Fletcher

[DESCRIPTION]
In this protocol, we aim to sequence multiple bacterial isolates from 1 contaminated food sample.  The goal is to obtain long-read sequences for food-borne pathogens to provide information regarding their virulence factors.  We will use Oxford Nanopore Technologies devices, kits, and protocols to prepare DNA libraries, sequence our bacterial samples, and analyze our sequencing data.

 


[STEPS]
SECTION: Part 1: Colony PCR DNA Extraction
1.1. Enriching for plasmid DNA only: If you are interested in the genomic DNA, proceed straight to step 2.3. If you are interested in the plasmid DNA, transfer the 50 μl cell suspension to a 0.2 ml thin-walled PCR tube and incubate at 95°C for 5 minutes.
Note: It is observed that pre-treating the colony by heating leads to an enrichment in the observation of plasmid reads in the downstream sequencing, compared with non-heat treated libraries.
SECTION: Part 1: Colony PCR DNA Extraction
1.2. Enriching for plasmid DNA only: Add 1 μl of Thermolabile Proteinase K and incubate at 37°C for 15 minutes, then 55°C for
10 minutes.
SECTION: Part 1: Colony PCR DNA Extraction
1. Because we are interested in genomic DNA for this protocol, obtain 1 colony from the
culture plate using a sterile toothpick, needle, or inoculation loop and swirl it in 50µL of 10mM Tris-HCl (pH: 8.0) for 10 seconds.
SECTION: Part 1: Colony PCR DNA Extraction
3. Repeat for however many individual colonies (i.e., bacterial samples) you wish to run (up to 24).
SECTION: Part 1: Colony PCR DNA Extraction
2. Libraries can be prepared for sequencing using the Rapid PCR Barcoding Kit, using 3 μl of the treated cell suspension as the template. If using cells without the heat treatment in step two, it is recommended to PCR for 25 cycles, and if using heat-treated cells, it is recommended to PCR for 30 cycles.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
4. .
 

SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
6. Adjust the volume to 3µL
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
7. Flick to mix and briefly spin down
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
8. Mix the following in a 0.2mL thin-walled PCR tube: 1-5ng template DNA (3µL), Fragmentation Mix (FRM) (1µL)
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
9. Flick to mix and briefly spin down
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
10. Incubate PCR tube in a thermal cycler at 30°C for 2 minutes, then 80°C for 2 minutes. a. Afterward, put the tube on ice briefly to cool down.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
11. For each sample, set up a PCR reaction in a 0.2mL thin-walled PCR tube as follows: a. Nuclease-free water (20µL) b. Tagmented DNA (4 µL) c. RLB Barcode (1-24, 1 µL) d. LongAmp Taq 2X Master Mix (25 µL)
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
12. Flick to mix and briefly spin down.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
13. Again in the thermal cycler, amplify DNA under the following conditions:
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
5. Transfer 1-5ng of each sample to a 1.5mL Eppendorf DNA LoBind tube.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
14. SS
 

SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
14.1. After PCR, hold at 4°C or continue
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
15. Add 4µL EDTA to each barcoded sample.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
16. Pipet to mix and briefly spin down.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
17. Incubate for 5 minutes at room temperature.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
18. Quantify 1 µL of each barcoded sample using a Qubit 4 Fluorometer.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
19. Pool all barcoded samples in equimolar ratios for a final concentration of 200-400fmol (~400-800ng) for pooled samples.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
20. Resuspend AMPure XP Beads (AXP) by vortexing.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
21. To the pool of barcoded samples, add a 0.6X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
22. Incubate on a Hula mixer for 5 minutes at room temperature.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
23. Prepare 2 mL of 80% ethanol in nuclease-free water.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
24. Briefly spin down.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
25. Pellet on magnet until supernatant is clear & colorless.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
26. Keeping tube on the magnetic rack, pipet off supernatant and discard
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
27. Keeping tube on magnetic rack, wash beads with 1 mL 80% ethanol without disturbing
pellet.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
27.1. Remove the ethanol using a pipet and discard.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
27.2. Repeat step 28.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
28. Spin down and place the tube back on the magnet. Pipet any residual ethanol and allow to dry for 30 seconds.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
29. Remove tube from magnetic rack and resuspend pellet in 15 µL elution buffer (EB).
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
30. Spin down and incubate for 5 minutes at room temperature.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
31. Pellet the beads on a magnet until supernatant is clear and colorless.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
31.1. . At least 1 minute.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
32. Remove and retain eluate into a sterile 1.5mL Eppendorf DNA LoBind tube.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
33. Quantify 1 µL using Qubit.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
34. Transfer 10–50 fmol of your eluted samples into a clean 1.5 mL Eppendorf DNA LoBind tube. Make up the volume to 11 µl with Elution Buffer (EB).
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
35. In a fresh 1.5 mL Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix: Rapid Adapter (RA, 1.5 µL), Adapter buffer (ADB, 3.5 µL)
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
36. Add 1 µL of the diluted Rapid Adapter (RA) to the barcoded DNA.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
37. Flick to mix and briefly spin down.
SECTION: Part 2: DNA Library Prep using ONT Rapid PCR Barcoding Kit
38. Incubate for 5 minutes at room temperature.
SECTION: Part 3: Load into MinION Flow Cell and Sequence Using GridION Sequencing Device
39. Load following manufacturer’s instructions, adding sample to SpotOn port in a drip-wise fashion.
SECTION: Part 3: Load into MinION Flow Cell and Sequence Using GridION Sequencing Device
40. Insert the MinION flow cell into the GridION device and sequence using MinKNOW with the correct DNA Library Prep Kit selected (Rapid PCR Barcoding Kit).
SECTION: Part 4: Analyze using EPI2ME Labs
41. Analyze the sequence data using the wf-metagenomics (NextFlow) and What’s in My Pot (WIMP, EPI2ME) workflows on the EPI2ME Labs software.
SECTION: Part 4: Analyze using EPI2ME Labs
42. Upload sample sheet with barcode IDs.
SECTION: Part 4: Analyze using EPI2ME Labs
43. Follow default settings.
SECTION: Part 4: Analyze using EPI2ME Labs
43.1. Decide if you want to gather antimicrobial resistance (AMR) data.