TITLE: Pan-microbial metagenomics protocol
AUTHORS: Adela Alcolea-Medina, Luke Blagdon Snell, Chris Alder, Rahul Batra

[DESCRIPTION]
DOI: 10.1038/s43856-024-00554-3


[STEPS]
SECTION: Preparation of quality controls:
2.  Mix 300μL of control 1 and 300μL of control 2 in an Eppendorf. Label with date and LOT number. 
SECTION: Preparation of quality controls:
3. Vortex for 1 minute and Centrifuge at 1,200xg for 5min immediately prior to each use.
SECTION: Preparation of quality controls:
4. Aliquot the volume specified by the manual or automated extraction method and process in parallel with equivalent clinical sample volumes.
SECTION: Preparation of quality controls:
5. Store the mixed positive controls at 4°C, it can be used for two extractions.
SECTION: Preparation of quality controls:
6.  Run a target PCR (Viasure respiratory panel III) anytime a new box is opened and record it on the trend analysis spreadsheet.
SECTION: Preparation of quality controls:
1. Positive control: NATtrol‱ Respiratory Panel 2.1 (RP2.1) Controls (Zeptometrix)
SECTION: Negative control: Respiratory Swab Matrix Negative Control (Vircell)
7. Add 500μL of Microbial DNA-Free Water to a vial and mix until completely reconstituted as per
manufacturer’s instructions. Vortex for 30 sec to dissolve and homogenise completely. Spike with 
1μL of TMV.
SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
8.  Reconstitute a new vial of TMV every Monday with 3mL of Microbial DNA-Free Water 
(Qiagen). Vortex for 1 min.
SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
9. Aliquot 500μL of IC into six Eppendorf tubes, labelling each tube with the preparation date and lot number. Store at 4°C.
SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
10. Use one aliquot per day to spike your samples and negative control, vortex for 1 min before spiking
SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
11. For every new vial reconstituted, TMV target PCR should be performed and Ct values should be recorded for trend analysis. 
- 200ul of neat was extracted directly from the vial (no depletion performed). 
 -In our hands the Ct value was 5-6
SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
12. Discard any remaining volume from used aliquot each day and any remaining aliquots by 
Friday.

SECTION: Internal control (IC): Tobacco Mosaic Virus (TMV) PC-0107 (DSMZ)
13. Spiking samples with the internal control (IC):

Spike samples with 1μLof TMV in the following samples:
-Broncho-alveolar lavage (BAL)
-Non-directed Broncho-alveolar lavage (NBL)
-Endotracheal tube (ETT) aspirates
-Sputum (SPT)

Spike with 0.5μL of TMV the following samples:
-Pleural fluids (PF)
-Nasal throat swabs (NTS)
SECTION: Sample preparation:
14. -Respiratory samples validated for this protocol are: BAL, NBL, ETT, NTS, sputum and PF. 
-Samples processed should be fresh up to four days old storage in the fridge, older than that it could 
not be guarantee the RNA virus detection.
- Samples are processed in a different way depending on the sample type.
SECTION: Sputum and ETT:
15. Mucoid samples such as sputum or ETT should be mucolysed before starting the Human DNA 
depletion process as follows:

Add approximately 2 mL sputum into a numbered, sterile, plastic universal container or if 2 mL 
already in original container then do the following: 
    
SECTION: Sputum and ETT:
16. Add equal volume (2mL) of mucolytic agent Mucolyse to the sample container and Vortex in the safety cabinet for 30 sec.
SECTION: Sputum and ETT:
17. Leave until liquefication at room temperature (RT).
SECTION: Sputum and ETT:
18. Gently agitate for a further 15 sec before to proceed to the human DNA depletion
SECTION: Sputum and ETT:
19. If after mucolysing the sample it is still mucoid, pipette 500μL of the sample and mix with 500μLof 
PBS, vortex well and proceed with the human DNA depletion. 
SECTION: Human DNA depletion:
20. The human DNA depletion is three steps: centrifugation, bead-beating and endonuclease treatment. 
However, BAL, NBL and ETT aspirates are processed in a different way depending on the appearance 
of the sample. This is due to the different composition of the sample.
SECTION: Human DNA depletion:
22. Transparent samples with some mucoid debris: the centrifugation step should be skipped and only bead-beating and endonuclease treatment should be performed.
SECTION: Human DNA depletion:
23. For clear samples with watery appearance: the human DNA depletion should be avoided and the sample should just be extracted.
SECTION: 1) Centrifugation:
24.  Vortex the sample     
SECTION: 1) Centrifugation:
26. Aliquot 500μL of the supernatant into an Eppendorf tube
If centrifugation is being removed then vortex sample well and add 500ul of whole sample. 
SECTION: 1) Centrifugation:
27. Spike 1μL of TMV to the sample (as indicated above) and 0.5μL for processing a PF and NTS. Make sure to vortex the TMV aliquot before use. 
SECTION: 1) Centrifugation:
28. Vortex the sample with the TMV 
SECTION: 1) Centrifugation:
29.  Procced with bead beating host-depletion step.
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
30. Transfer sample from Eppendorf (500ul) to the Matrix 
Lysing D tube (MP biomedicalsTM) after vortexing for 1 min. 
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
31. Place the matrix tube into the TissueLyser LT (Qiagen). Bead-beat at 50 osc for 3min. 
Note: sometimes the sample will become foamy after bead-beating, this is ok.
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
32. Transfer 200μL of the bead-beaten sample to a new Eppendorf tube, placing the tip at the 
bottom of the tube to take-up the liquid instead of the foam.
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
33. Add 10μL of HL-SAN enzyme (ArticZymes Technology). 
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
34. Mix for 10’ at 37⁰C at 1000rpm in the Eppendorf ThermoMixer (EppendorfTM).
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
35. NOTE: The HL-SAN is stopped by the Magnapure extraction when the instrument adds 
proteinase k. However, not all the extraction instruments/kits add proteinase k. If the 
extraction instrument/kit does not have this step, add the proteinase K manually as 
followed:
 
-Add 10μL of proteinase K to 200ul of the samples previously treated with HL-SAN.
-Vortex for 10 sec.
-Incubate at 56⁰C for 10 min at 1000rpm.
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
36. Transfer 200μL to Magnapure 24 (RocheTM) for extraction or proceed using alternative 
extraction methods validated in your own laboratory
SECTION: 2) Bead beating and depletion of free nucleic acid, followed by extraction:
37. Use total nucleic acid extraction kit 1.1 with pre-set BAL sample parameters and 50μL 
elution volume.

 Fast pathogen 200 1.1 is used for processing <8 samples 

 Pathogen 200 3.2 for >=8 samples. Settings for other extraction robots will need to be established in user 
laboratories.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
38.  RT-dsDNA:
SECTION: 1. Microbial nucleic acid processing and PCR amplification
41. Place the tube in the thermocycler and incubate at 25⁰C for 2 min, 55⁰C for 10 
min, 95⁰C for 1 min.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
42.  Prepare the master mix for the double strand synthesis using the Sequenase Version 2.0 
DNA Polymerase using the following volumes: 

 

SECTION: 1. Microbial nucleic acid processing and PCR amplification
44. Place the tube in the thermocycler. Incubation at 37⁰C for 8 min.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
45.  Prepare stock of 70% (700μL absolute ethanol+ 300μL of Microbial DNA-free water for 
1mL) ethanol in Microbial DNA-Free Water.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
46.  Add 45μL of resuspended AMPure XP Beads (AXP) into a new Eppendorf tube for each 
sample.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
47. Add all of the product post dsDNA synthesis (31.2μL) into the Eppendorf tube containing 
the beads, mix well by flicking the tube and incubate at RT for 5 min. 
SECTION: 1. Microbial nucleic acid processing and PCR amplification
43.  Add 11.2μL  of the master mix to the 20μL of product post RT step. Mix by flicking the 
tube and briefly spin down.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
48. Briefly spin down the samples and pellet the beads on a magnet until the eluate is clear 
and colourless. Keep the tubes on the magnet and discard the supernatant.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
49. Keep the tubes on the magnet and wash the beads with 200μL of freshly prepared 70% 
ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
50. Repeat the previous step. 
SECTION: 1. Microbial nucleic acid processing and PCR amplification
51. Briefly spin down and place the tubes back on the magnet. Pipette off any residual 
ethanol with the 10p pipette. Allow to dry for 30 sec, but do not dry the pellet to the 
point of cracking.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
52. Remove the tubes from the magnetic rack and resuspend the pellet in 12μL of Microbial 
DNA free water. Spin down and incubate for 2 min at RT.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
53.  Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: 1. Microbial nucleic acid processing and PCR amplification
54. Remove and retain 10μL of eluate for each sample into clean 1.5 mL Eppendorf DNA 
LoBind tubes, individually.
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
56. Add 3μL of the previous product (dsDNA) to a new PCR tube.     
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
58. Place the tube in the thermocycler and incubate at 30⁰C for 1 min and then 80⁰C for 1 
min.
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
59. Prepare the master mix for the PCR, adding 20μL of water and 25μL of LongAmp Taq 2X 
Master Mix per sample and 1μL of the barcode primers to 4μL of the product obtained after 
the FRM step.
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
60.  Mix by gently  flicking the tube and briefly spin down.
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
61.  Place in the thermocycler. Incubate @ 95⁰ 3’, (95⁰ 15’’, 56⁰ 15’’, 65⁰ 4’) x30, 65⁰ 4’
SECTION: Post PCR:
62. Perform Qubit‱ 1X dsDNA High Sensitivity (HS) on the PCR products as the 
manufacturer’s instructions.
SECTION: Post PCR:
63. 
 

SECTION: Post PCR:
64. Pool samples as follows:
Based on the qubit readings, prioritise samples with a qubit reading below 1 ng/µL. For 
these samples, normalise the qubit concentration of your PCR products to 100 ng/µL.
n=sample 
e.g. 100/(n)0.6= 166.67 add the total volume of PCR product.
SECTION: Post PCR:
65. Clean up 1:1 by adding the same volume of resuspended AMPure XP Beads (AXP) to the same volume of the pooled samples. for example, 50ul of pool = 50ul of beads
SECTION: Post PCR:
66. Mix well by flicking the tube, incubate for 5 min in the Hula mixer (rotor mixer) at 
RT.
SECTION: Post PCR:
67. Briefly spin down the samples and pellet the beads on a magnet until the eluate is clear 
and colourless. Keep the tubes on the magnet and discard the supernatant.
SECTION: Post PCR:
68. Keep the tubes on the magnet and wash the beads with 200μL of freshly prepared 70% 
ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

 Repeat the previous step.
SECTION: Post PCR:
69. Briefly spin down and place the tubes back on the magnet. Pipette off any residual 
ethanol with the 10p pipette. Allow to dry for 30 sec, but do not dry the pellet to the 
point of cracking.
SECTION: Post PCR:
70. Remove the tubes from the magnetic rack and resuspend the pellet in 15μL Microbial 
DNA-Free Trish buffer. Spin down and incubate for 2 min at RT.
SECTION: Post PCR:
71. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: Post PCR:
72. Remove and retain 10μL of eluate into clean 1.5 mL Eppendorf DNA LoBind tubes, 
individually. Make sure no beads are remaining. 
SECTION: Post PCR:
73. -Add RAP 1μLto 10μL of template, mix gently by flicking the tube, spin down and incubate 
at RT for 5 min.
- Add 25.5μL of LB 
- Add 34μL of SQB
- Add 4.5μL of water
- Mix gently pipetting up and down 
SECTION: Flowcell loading:
74.  Thaw FLB and FLT at RT if not thawed from before. 
SECTION: Flowcell loading:
75. Add 30μL of FLT (vortex and spin before by pipetting) to a FLB (vortex before by pipetting).  Mix by vortex. 
SECTION: Flowcell loading:
76. Open the GridION device lid and slide the flow cell under the clip. Press down firmly on 
the flow cell to ensure correct thermal and electrical contact.
SECTION: Flowcell loading:
77. Slide the flow cell priming port cover clockwise to open the priming port.
SECTION: Flowcell loading:
78. After opening the priming port, check for a small air bubble under the cover. Draw back 
a small volume to remove any bubbles. 
SECTION: Flowcell loading:
79.  Set a P1000 pipette to 200μL and insert the tip into the priming port. 
SECTION: Flowcell loading:
80.  Turn the wheel until the dial shows 220-230μL , to draw back 20-30μL , or until you can 
see a small volume of buffer entering the pipette tip 

Note: Visually check that there is continuous buffer from the priming port across the sensor array.
SECTION: Flowcell loading:
81.  Load 800μL of the priming mix into the flow cell via the priming port, avoiding the 
introduction of air bubbles. Wait for five min. 
SECTION: Flowcell loading:
82.  Gently lift the SpotON sample port cover to make the SpotON sample port accessible. 
SECTION: Flowcell loading:
83. Load 200μL of the priming mix into the flow cell priming port (not the SpotON sample 
port), avoiding the introduction of air bubbles.
SECTION: Flowcell loading:
84. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Flowcell loading:
85.  Add 75μL of the prepared library 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: Flowcell loading:
86. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON 
port, close the priming port and replace the MinION or GridION device lid.
SECTION: GridION settings:
87. Select the position of your flow cell on MinKNOW.
SECTION: GridION settings:
88. Type your experiment name and sample ID.
SECTION: GridION settings:
89. Continue to kit selection and select Rapid PCR barcoding kit SQK -RPB004.
SECTION: GridION settings:
90. Continue to run options and change to 24hr.
SECTION: GridION settings:
91. Continue to analysis and edit options and select barcode both ends and mid read barcode filtering.
SECTION: GridION settings:
92. Continue to output and unselect fast 5 and edit FastQ to 100 reads per file.
SECTION: Bioinformatic pipeline and reporting:
93. Curated bioinformatic pipeline will generate automatic reports at 30 min (for organism 
identification) and 2 hr (for AMR determinants). 16-24 hr reports can be generated 
manually for SNP typing and genomic alignment.
SECTION: qPCR for the TMV
95. The RT step is performed using the LunaScript master mix explained previously in the step 39. 

SECTION: qPCR for the TMV
96. Master-mix calculations as followed:
 
1094/PHYTO-06-19-0201-FI. Epub 2019 Nov 18. PMID: 31502520.

Primer sequence for TMV: 
-TMV_fwd_aps:
Catalog number: 10336022
GGATATGTCTAAGTCTGTTGC 
10629186 Nucleotides (25 nmole) 11732013 Desalted. 

-TMV_rvr_aps:
Catalog number: 10336022
CAGACAACTCGGGTGCG 
10629186 Nucleotides (25 nmole) 11732013 Desalted.



SECTION: qPCR for the TMV
97. 1 cycle at 95°C for 20 s, 40 cycles at 95°C for 1 s, 40 cycles at 60°C for 20 s, 1 cycle at 95°C for 15 s, 1 
cycle at 60°C for 1 min, and 1 cycle at 95°C for 15 s
SECTION: Human DNA depletion:
21. For turbid and mucoid samples: the human DNA depletion is performed as usual, centrifugation followed by bead-beating and endonuclease treatment.
SECTION: 1) Centrifugation:
25. Centrifuge speed 1,200xg for 10 min. 
    
SECTION: 1. Microbial nucleic acid processing and PCR amplification
39. Transfer 16μL of the 50μL  extract (from Magnapure) to a PCR tube and add 4μL of
LunaScript‱ RT SuperMix.     
SECTION: 1. Microbial nucleic acid processing and PCR amplification
40. Mix by flicking the tube and briefly spinning down in a small bench 
top Eppendorf centrifuge. 
SECTION: 1. Microbial nucleic acid processing and PCR amplification
55. NOTE: the sample can be store at -80°C after the RT or dsDNA step
SECTION: 2. Library preparation Rapid PCR Barcoding Kit (SQK-RPB004) Oxford Nanopore:
57. Add 1μL of FRM (from ONT Rapid PCR barcoding kit SQK -RPB004). Mix by flicking the tube and briefly spin down.
SECTION: Bioinformatic pipeline and reporting:
94. This is the link for the bioinformatic pipeline analysis GitHub - GSTT-CIDR/RespiratoryCmg . 
NOTE: A new version will be available soon.
SECTION: qPCR for the TMV
98. The validation of this method is available on Unified metagenomic method for rapid detection of microorganisms in clinical samples | Communications Medicine (nature.com).