TITLE: Characterization of the Archaeome, Bacteriome and Eukaryome in Nasopharyngeal Swabs
AUTHORS: Carolin Baehren, Anton  Pembaur, Patrick P. Weil, Frank Schult, Stefan Wirth, Jan Postberg, Malik Aydin

[DESCRIPTION]
This protocol describes the Characterization of the Archaeome, Bacteriome and Eukaryome in Nasopharyngeal Swabs by sequencing with nanopore technology.

For a long time, archaea were under-represented in the literature, and less is known about their pathogenicity in human diseases. Using conventional methods, the cultivability particularly of archaea is challenging and they are still classified as the ‘dark matter’ of the microbiome. The evolution of advanced sequencing techniques in the twenty-first century, a strong focus on archaea research is interestingly observed. However, the influence on disease course or even pathogenesis in terms of respiratory disorders remain unexplored. Thus, more attention has to be paid on the characterization of the archaeome with the goal of translation into clinical contexts. Considering this important issues lacking good methodological reports in the literature, we evaluated previously developed primer sets and sequencing platforms. With these useful hints, we share potential alternative procedures with the aim how to increase the quality of research on archaeome and eukaryotes. The use of nasopharyngeal swab specimens derived from a cohort suffering from respiratory diseases enable to study translational aspects on disease course and eventually pathogenesis. The optimization of ‘pre-sequencing’ steps, starting from the DNA isolation, amplification, right choice of sequencing platforms e.g., MinION Oxford Nanopore rule some important traces to a high-qualitative in-depth sequencing success. However, those descriptive data significantly contribute to optimize existing archaic models with the aim to exploit translational approaches ex vivo. 

[STEPS]
SECTION: PCR
2. PCR 
Archaea: Nested PCR
Eukaryotes: single PCR
SECTION: Library preparation + sequencing:
3. Library Preparation + Sequencing:

- 1st Purification
- PCR preparation
- 2nd Purification
- Concentration measurement 
SECTION: Bioinformatics:
4. 




SECTION: DNA Isolation
1.2. Resuspension of pellet in 180 µL

SECTION: DNA Isolation
1.1. Centrifugation of sample at 7500 rpm, 10 min  until pellet formation.

SECTION: DNA Isolation
1. DNA was isolated from nasal swabs with amies medium, using approximately 1000 µL .
For the Isolation, the QIAmp DNA-Mini Kit by Qiagen was used, following the QiAamp tissue protocol from the ‘QIAamp DNA Mini and Blood Mini Handbook 05/2016’

SECTION: DNA Isolation
1.3. Adding 20 µL, vortexing, 
Incubate at 56 °C until complete lysis. Occasionally vortexing.

SECTION: DNA Isolation
1.4. Brief Centrifugation of the sample.
SECTION: DNA Isolation
1.5. Adding 200 µL, pulse-vortex afterwards 15 s, Incubation at 70 °C for 10 min . Brief centrifugation of the sample.

SECTION: DNA Isolation
1.6. Adding 200 µL, and mix by pulse-vortexing for 15 s. Afterwards, short centrifugation of the sample.
SECTION: DNA Isolation
1.7. Transfer mixture (including precipitate) to the QIAamp Mini spin column. CAVE: without wetting the rim. Centrifugation: 8000 rpm, 1 min
Replace the QIAamp Mini spin column, use a clean 2 ml collection tube, Discard tube with the filtrate.

SECTION: DNA Isolation
1.8. Adding 500 µL CAVE: without wetting the rim. 
Centrifuge:8000 rpm, 1 min. 
Replace the QIAamp Mini spin column, use a clean 2 ml collection tube, Discard tube with the filtrate.

SECTION: DNA Isolation
1.9. Add 500 µL to the QIAamp Mini spin column without wetting the rim. Closing of the column, Centrifugation: 14000 rpm, 3 min

SECTION: DNA Isolation
1.10. Replace QIAamp Mini spin column with a new 2 ml collection tube. Discard tube with the filtrate. Centrifugation: 14000 rpm, 1 min 

SECTION: DNA Isolation
1.11. Placing QIAamp Mini spin column in a new 1.5 ml microcentrifuge tube. Discard tube with the filtrate. 
Add 100 µL. Incubation at room temperature (5 min), centrifugation:8000 rpm, 1 min 

SECTION: DNA Isolation
1.12. Repeat Step 1.11: Add the flowthrough of the previous step to the Mini spin column and incubation at room temperature for 5 min, centrifugation 8000 rpm, 1 minat .


SECTION: DNA Isolation
1.13. Concentration measurement with nanophotometer or qubit.
SECTION: PCR
2.1. Primer selection for archaea and eukaryotes
 
Nr.NamePrimer NameSequence (5’  3’)1344FS-D-Arch-0344-fw5’-acggggygcagcaggcgcga-3’21041RS-D-Arch-1041-rev5’-ggccatgcaccwcctctc-3’3519FArch-519F-Tag5’-tttctgttggtgctgatattgccagcmgccgcggtaa-3’4786RArch-786R-Tag5’-acttgcctgtcgctctatcctcggactacvsgggtatctaat-3’5563FEuk-563F-Tag5’-tttctgttggtgctgatattgcgccagcavcygcggtaay-3’61132REuk-1132R-Tag5’-acttgcctgtcgctctatcttcccgtcaatthcttyaart-3’
 
SECTION: PCR
2.2. 1st PCR Mix:
8 µL
12.5 µL
2 µL
2.5 µL

SECTION: PCR
2.3. PCR-Run.    1
Primer pair Arch-344-F-1041R / Eck.563F-1132Rtag

Heated Lid: 110 C
Denaturation	        95 °C 3 min
Cycles (30):
Denaturation        95 °C 30 s
Annealing	       55 °C 30 s
Elongation             72 °C30 s
End Cycle
Final Elongation	65 °C5 min
SECTION: PCR
2.4. 2nd PCR (archaea only) Mix:
9.5 µL
12.5 µL
2 µL
1 µL

SECTION: PCR
2.5. PCR-Run.    2 (nested)
Primer pair Arch-519-F-786Rtag 

Heated Lid: 110 C
Denaturation    	95 °C 3 min
Cycles (28):
Denaturation        95 °C 30 s
Annealing	        55 °C 30 s
Elongation 	        72 °C30 s
End Cycle
Final Elongation	65 °C5 min
2.6. Gel Electrophoresis

Check, if the wanted sequences were amplified. Ether through classic gel electrophoresis or through microcapillary gel electrophoresis.
SECTION: Library preparation + sequencing:
3.1. 1st Purification:
Add 36 µL  and apply an external magnetic field for 5 min. Afterwards discard fluid supernatant.
SECTION: Library preparation + sequencing:
3.6. 
Heated Lid: 110 C
Denaturation	         95 °C3 min
Cycles (18):
Denaturation         95 °C15 s
Annealing	        62 °C15 s
Elongation 	        65 °C45 s
End Cycle
Final Elongation	65 °C5 min


SECTION: Library preparation + sequencing:
3.8. Add 36 µL and apply an external magnetic field for 5 min. Afterwards discard fluid supernatant

SECTION: Library preparation + sequencing:
3.7. 2nd Purification

SECTION: Library preparation + sequencing:
3.9. Add 150 µL and discard fluid supernatant.

SECTION: Library preparation + sequencing:
3.10. Add another  150 µL . Afterwards discard the fluid supernatant and dry tube with open lid

SECTION: Library preparation + sequencing:
3.11. Resuspend pellet in 15 µL
SECTION: Library preparation + sequencing:
3.12. Concentration measurement with nanophotometer 
SECTION: Library preparation + sequencing:
3.13. Library preparation:

We used a modified version of the PCR barcoding (96) genomic DNA (SQK-LSK109) protocol by Nanopore.
Quantify the barcoded library using a nanophotometer and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.
SECTION: Library preparation + sequencing:
3.2. Add 150 µL and discard fluid supernatant.
SECTION: Library preparation + sequencing:
3.3. Add another 150 µL . Afterwards discard the fluid supernatant and dry tube with open lid

SECTION: Library preparation + sequencing:
3.4. Resuspend pellet in 15 µL

SECTION: Library preparation + sequencing:
3.5. PCR-preparation: Mix

12.5 µL 
2 µL
9.5 µL
1 µL 

SECTION: Library preparation + sequencing:
3.14. Prepare 1 µg  in 47 µL.

SECTION: Library preparation + sequencing:
3.15. DNA repair and end-prep 
SECTION: Library preparation + sequencing:
3.33. Adapter ligation and clean-up (PCR barcoding (96) genomic DNA (SQK-LSK109) protocol by Nanopore)

Although the recommended 3rd party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.
SECTION: Library preparation + sequencing:
3.51. Priming and loading the SpotON flow cell 
SECTION: Library preparation + sequencing:
3.16. Thaw DNA CS (DCS) at RT, spin down, mix by pipetting, and place on ice.
SECTION: Library preparation + sequencing:
3.17. Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
SECTION: Library preparation + sequencing:
3.18. In a 0.2 ml thin-walled PCR tube, mix the following: 

1 µL
47 µL
3.5 µL
2 µL
3.5 µL
3 µL

Mix gently by flicking the tube, and spin down.
SECTION: Library preparation + sequencing:
3.19. Using a thermal cycler, incubate at 20 °C for 5 min and 65 °C for 5 min

SECTION: Library preparation + sequencing:
3.20. AMPure XP bead clean-up
SECTION: Library preparation + sequencing:
3.21. Resuspend the AMPure XP beads by vortexing. 
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Library preparation + sequencing:
3.22. Add 60 µL to the end-prep reaction and mix by flicking the tube.

SECTION: Library preparation + sequencing:
3.23. Incubate on a Hula mixer (rotator mixer) for 5 min at room temperature.

SECTION: Library preparation + sequencing:
3.24. Prepare 500 µL in Nuclease-free water.

SECTION: Library preparation + sequencing:
3.25. Spin down the sample and pellet on a magnet until eluate is clear and colourless. 
Keep the tube on the magnet, and pipette off the supernatant.
SECTION: Library preparation + sequencing:
3.26. Keep the tube on the magnet and wash the beads with200 µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Library preparation + sequencing:
3.27. Repeat the previous step.
SECTION: Library preparation + sequencing:
3.28. Spin down and place the tube back on the magnet. 
Pipette off any residual ethanol. Allow to dry for 30 s, but do not dry the pellet to the point of cracking.

SECTION: Library preparation + sequencing:
3.29. Remove the tube from the magnetic rack and resuspend the pellet in 61 µL
Incubate for 2 min at RT.

SECTION: Library preparation + sequencing:
3.30. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: Library preparation + sequencing:
3.31. Remove and retain 61 µL into a clean 1.5 ml Eppendorf DNA LoBind tube. 

SECTION: Library preparation + sequencing:
3.32. Take forward the repaired and end-prepped DNA into the adapter ligation step. 
However, at this point it is also possible to store the sample at 4 °C overnight.

SECTION: Library preparation + sequencing:
3.34. Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.
SECTION: Library preparation + sequencing:
3.35. Thaw Ligation Buffer (LNB) at RT, spin down and mix by pipetting. 
Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
SECTION: Library preparation + sequencing:
3.36. Thaw the Elution Buffer (EB) at RT, mix by vortexing, spin down and place on ice.
SECTION: Library preparation + sequencing:
3.37. To retain DNA fragments of < 3 KB, thaw one tube of Short Fragment Buffer (SFB) at RT, mix by vortexing, spin down and place on ice.
SECTION: Library preparation + sequencing:
3.38.  In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
 
60 µL
25 µL
10 µL
5 µL

 Mix gently by flicking the tube, and spin down.
 
SECTION: Library preparation + sequencing:
3.39. Incubate the reaction for 10 min at RT. If you have omitted the AMPure purification step after DNA repair and end-prep, do not incubate the reaction for longer than 10 min.

SECTION: Library preparation + sequencing:
3.40. Resuspend the AMPure XP beads by vortexing. Add 40 µL to the reaction and mix by flicking the tube.


SECTION: Library preparation + sequencing:
3.41. Incubate on a Hula mixer (rotator mixer) for 5 minat RT.

SECTION: Library preparation + sequencing:
3.42. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: Library preparation + sequencing:
3.43. Wash the beads by adding  250 µL. 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.

SECTION: Library preparation + sequencing:
3.44. Repeat the previous step.
SECTION: Library preparation + sequencing:
3.45. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 30 sbut do not dry the pellet to the point of cracking.

SECTION: Library preparation + sequencing:
3.46. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL. Spin down and incubate for 10 min at RT. 

SECTION: Library preparation + sequencing:
3.47. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: Library preparation + sequencing:
3.48. Remove and retain 15 µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Library preparation + sequencing:
3.49. Quantify 1 µL using a Qubit fluorometer. The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

SECTION: Library preparation + sequencing:
3.50. The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.
SECTION: Library preparation + sequencing:
3.52. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at RT.
SECTION: Library preparation + sequencing:
3.53. Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by vortexing and spin down at RT.
SECTION: Library preparation + sequencing:
3.54. Open the MinION Mk1B lid and slide the flow cell under the clip. Slide the priming port cover clockwise to open the priming port.

Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
SECTION: Library preparation + sequencing:
3.55. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):

Set a P1000 pipette to 200 μl
Insert the tip into the priming port
Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip
SECTION: Library preparation + sequencing:
3.56. To prepare the flow cell priming mix, add 30 µL directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at RT.

SECTION: Library preparation + sequencing:
3.57. Load 800 µL mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min. During this time, prepare the library for loading by following the steps below.

SECTION: Library preparation + sequencing:
3.58. Thoroughly mix the contents of the Loading Beads (LB) by pipetting because it contains a suspension of beads which settle very quickly. It is vital that they are mixed immediately before use!
SECTION: Library preparation + sequencing:
3.59. In a new tube, prepare the library for loading as follows:
37.5 µL
25.5 µL, mixed immediately before use 
12 µL

SECTION: Library preparation + sequencing:
3.60. Complete the flow cell priming through Gently lifting the SpotON sample port cover to make the SpotON sample port accessible. Load 200 µL into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

SECTION: Library preparation + sequencing:
3.61. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Library preparation + sequencing:
3.62. Add 75 µL to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

SECTION: Library preparation + sequencing:
3.63. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the lid.
SECTION: Library preparation + sequencing:
3.65. Ending the experiment

After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit instructions and store the washed flow cell at 2-8°C, OR

Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.
SECTION: Library preparation + sequencing:
3.64. If you using a MinION Mk1C  turn basecalling while sequencing on.
SECTION: Bioinformatics:
4.1. If you were unable to basecall in real time, perform the basecalling now using the Guppy basecaller (newest version).

https://community.nanoporetech.com/docs/prepare/library_prep_protocols/Guppy-protocol/v/gpb_2003_v1_revag_14dec2018/guppy-software-overview
SECTION: Bioinformatics:
4.2. Now, using the resulting .fastq files, run the WIMP workflow from the Epi2Me software.

(https://nanoporetech.com/resource-centre/epi2me-wimp-workflow-quantitative-real-time-species-identification-metagenomic)
SECTION: Bioinformatics:
4.3. If the graphical output from the WIMP workflow is not sufficient for your analysis, you can download the results in a .csv dataset. Due to the size of this dataset,  further analyses may be performed by creating an SQL database.

The data contains the 
filename of the .fastq file
Read ID --> is the unique primary key, wich enables to identify the read and therefore the sequence
Run ID
exit_status (of the read from the WIMP workflow)
barcode
taxID (every phylogenetic rank of each species has its own ID, with these IDs the lineage is composed
name (of the organism)
score
lineage
SECTION: Bioinformatics:
4.4. Python scripts

While working on this project, a few Python scripts may be useful, depending on analysis you want to perform.


This script we used to split large files into smaller ones:

 


This script was used, to append the lenght of each analysed read (or with small changes the whole sequence) to the .csv table: