Unbiased Metagenomic Protocol: From RNA to Sequencing

Alexander Martínez; Claudia González; Shannon Whitmer; Oris Chavarría; Jessica Góndola; Ambar Moreno

May 05, 2025

Unbiased Metagenomic Protocol: From RNA to Sequencing

DOI

dx.doi.org/10.17504/protocols.io.36wgq6545lk5/v1

Alexander Martínez^1,2,3,
Claudia González^1,2,
Shannon Whitmer⁴,
Oris Chavarría¹,
Jessica Góndola¹,
Ambar Moreno¹

¹Department of Research in Genomics and Proteomics, Gorgas Memorial Institute for Health Studies, Panama;
²Department of Microbiology and Immunology, University of Panama, Panama;
³Sistema Nacional de Investigación, SENACYT, Panama City, Republic of Panama;
⁴Viral Special Pathogen Branch, U.S Centers for Disease Control and Prevention (CDC), Atlanta GA, USA

Alexander Martinez

Gorgas Memmorial Institute for Health Studies

DOI: dx.doi.org/10.17504/protocols.io.36wgq6545lk5/v1

Protocol Citation: Alexander Martínez, Claudia González, Shannon Whitmer, Oris Chavarría, Jessica Góndola, Ambar Moreno 2025. Unbiased Metagenomic Protocol: From RNA to Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq6545lk5/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and have successfully sequenced viruses such as Oropouche, Respiratory Syncytial Virus, Equine Encephalitis Virus, Rhinovirus, Madariaga, and Influenza.

Created: March 25, 2025

Last Modified: May 05, 2025

Protocol Integer ID: 125642

Keywords: Metagenomics, DNA, RNA, Non-targeted sequencing

Funders Acknowledgements:

Ministerio de Economía y Finanzas Panamá

Grant ID: 19911.031

This publication was in part supported by the Cooperative Agreement Number NU50CK000639 awarded to the Pan American Health Organization and funded by the Centers for Disease Control and Prevention. ts contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention or the Department of Health and Human Services.

Grant ID: NU50CK000639

Disclaimer

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Abstract

To identify microorganisms in clinical samples with a diverse community through non-targeted sequencing, a de novo or shotgun sequencing process is proposed, starting from RNA obtained from clinical samples such as nasal swabs, serum, or viral culture isolates. Circulating DNA will be removed enzymatically, then converting the available RNA to cDNA -> dsDNA -> and then amplifying dsDNA. The obtained material can be sequenced by preparing a library with kit Nextera XT for Illumina platforms.

Guidelines

Use cooling blocks to maintain reagents during PCR steps.

Materials

Reagents

Qubit 1X dsDNA High Sensitivity (HS) - Invitrogen™ Cat. Q3323 
Qubit RNA High Sensitivity (optional) - Invitrogen™ Cat. Q32852 
TURBO DNA-free™ Kit - Ambion Life Technologies Cat. AM1907 
Nuclease-free water Cat AM9937 
10X Turbo DNase buffer.  
Turbo DNase, 2U/ μL. 
DNase Inactivation Reagent. 
3M Sodium acetate CAT S7899-500ML 
2-propanol 
Absolute ethanol molecular grade 
70% Ethanol.  
10nM dNTPs. 
Random Hexamers. 
Nuclease-free water. 
0.1M DTT. 
RNase Inhibitor 40 U/ μL. 
Superscript IV RT CAT 18090010. 
Superscript IV First-Strand Synthesis System - Invitrogen Cat. 18091050 
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - Cat. E6111S 
Agencourt AMPure XP - Cat. A63881 
GenomiPhi V2 DNA Amplification Kit - Cytiva Cat. 25660031 
Nextera XT sample prep Kit 96 Box 1 (-25°C to -15°C) CAT FC-131-1096 
Nextera XT library prep kit 96 sample Box 2 (4°C)  
Index adapters 96 samples: CAT FC-131-2001, CAT FC-131-2002, CAT FC-131-2003 and CAT FC-131-2004 
PhiX Control 10nM CAT FC-110-3001 
Qubit 1X dsDNA High Sensitivity (HS) - Invitrogen‱ CAT. Q3323 
1N NaOH CAT 06203-1KG

Equipments 

Thermal cycler 
Level II biosafety cabinet 
Vortex 
Centrifuge 
Microcentrifuge 
Plate centrifuge 
Plate shaker 
Magnetic rack for 1.5 ml microtubes CAT 12321D  
Fluorometer – Qubit 
Fragment analyzer 

ReagentQubit 1X dsDNA BR Assay KitInvitrogen - Thermo FisherCatalog #Q33230  
ReagentQubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852  
ReagentTURBO DNA-free™ KitThermo ScientificCatalog #AM1907  
ReagentNuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificCatalog #AM9937  
ReagentSodium Acetate buffer solution 3 M pH 52 for molecular biologyMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7899-500ML 
ReagentSuperScript&trade; IV Reverse TranscriptaseThermo FisherCatalog #18090010 
ReagentSuperScript™ IV First-Strand Synthesis SystemThermo Fisher ScientificCatalog #18091050 
ReagentNEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - 20 rxnsNew England BiolabsCatalog #E6111S 
ReagentAgencourt AMPure XP beadsBeckman CoulterCatalog #A63881 
ReagentGenomiPhi V2 DNA Amplification KitCytviaCatalog #25660031 
ReagentNextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096 
ReagentNextera XT Index Kit v2 (set A B C D)Illumina, Inc.Catalog #FC-131-2001; FC-131-2002; FC-131 , 
ReagentphiX V3 controlIllumina, Inc.Catalog #FC-110-3001 
ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230 
ReagentInvitrogen™ DynaMag™-2 MagnetThermo Fisher ScientificCatalog #12321D 

Safety warnings

Due to the unbiased amplification performed, follow a unidirectional workflow to avoid cross-contamination.

Ethics statement

This a research use only protocol, an approval by the users' Institutional Review Board (IRB) or equivalent ethics committee(s) must be obtained for the use on human or animal samples.

Before start

It is recommended to use freshly extracted nucleic acids or those stored for no longer than 3 days at Temperature-80 °C  . Consider the target (organisms, in case of a known suspect) you wish to obtain to determine the starting point of the process. If it is RNA, complete the entire process; if it is DNA, purification is recommended, followed by the GenomiPhi kit’s amplification step.

Nucleic Acid Quantification (Optional)

Follow the nucleic acid quantification procedure (RNA) indicated by the manufacturer for the Qubit fluorometer or the method available in your laboratory for RNA quantification.

If identifying microorganisms whose genetic material is DNA is required, quantify the initial material in the sample under study, perform purification with beads at a 1:1 ratio, and then proceed with the Amplification step.

Removal of Contaminating DNA from RNA Samples

35m

Mix Amount5 µL   of 10X Turbo DNase Buffer, Amount1 µL   of TURBO DNase (2U), and Amount4 µL   of nuclease-free water in a tube.

Dispense Amount10 µL   of the mix into a microtube/8-tube strip or a plate, depending on the number of samples to be processed.

Add RNA (~Amount40 µL  ) or Amount10 µg   of RNA per reaction, with a total reaction volume of Amount50 µL  , and mix gently.

Incubate at Temperature37 °C   for Duration00:30:00  .

30m

Mix the DNase Inactivation Reagent by pipetting or gently swirling the tube. 
Note
This reagent has a milky appearance; ensure it is homogeneous before use.

Add Amount3 µL   of DNase Inactivation Reagent to each well containing the samples at TemperatureRoom temperature   .

Incubate for Duration00:05:00   at TemperatureRoom temperature  , occasionally mixing by pipetting during the incubation.

Individually transfer the contents of the wells into a 1.5 mL microtube labeled with the sample ID for the next step.

RNA Precipitation

39m

Complete the volume of your DNase-treated RNA solution to a total of Amount500 µL   with nuclease-free water.

Add Amount50 µL   of Concentration3 Molarity (M)   sodium acetate at TemperatureRoom temperature   (pH 5.2-5.5).

Add Amount500 µL   of 2-propanol at TemperatureRoom temperature   and mix by inverting about 10 times to ensure homogeneity.

Let stand at TemperatureRoom temperature   for Duration00:20:00  .

20m

Centrifuge at Centrifigation12000 rpm, Room temperature, 00:15:00  to precipitate the RNA pellet.

15m

Be aware of which side of the tube the pellet is located.

Carefully remove the supernatant. 
Note
The sediment will appear transparent and glassy after the supernatant is removed. 
Be careful not to dislodge or remove the pellet with the pipette tip.

Add Amount500 µL   of chilled 70% ethanol to wash the sediment (1/2).

Centrifuge at Centrifigation12000 rpm, Room temperature, 00:02:00  .

Remove the supernatant. 
Note
Be very careful not to discard the pellet, which will appear white and more visible after the ethanol wash.

Wash with Amount500 µL   of chilled 70% ethanol (2/2).

Centrifuge at  Centrifigation12000 rpm, Room temperature, 00:02:00  .

Carefully remove all the remaining ethanol.

Leave the microtube open at TemperatureRoom temperature   to allow all the ethanol to evaporate, and ensure the pellet appears matte and completely white. 
Note
This step may take 5 to 30 minutes.

Resuspend the sediment in nuclease-free water in an appropriate volume (recommended: Amount30 µL  ).
Note
Note 1: Optionally, you may assess the RNA quantity and integrity using NanoDrop or the Fragment Analyzer.

First-Strand Synthesis

1h 41m

Note
Note 2: Consider processing each sample in duplicate. Consider serving reagents in a nucleic acid-free cabinet.
For the denaturation mix, add Amount1 µL   of Concentration10 millimolar (mM)   dNTPs, and Amount2 µL   of random hexamers, and dispense Amount3 µL   of the mix per reaction into a 96-well plate/tube according to the number of samples to be processed.

For reverse transcription, mix the following reagents:  
AB
water2 μL
SSIV buffer 4 μL
0.1 M DTT1 μL
RNase inhibitor (40 U/μL)1 μL
SuperScript IV enzyme1 μL
 

Gently mix the RNA and then perform a short centrifugation to remove droplets from the lid. 
Dispense Amount12 µL   of RNA into each well containing the denaturation mix, briefly centrifuge, and place in the thermal cycler at Temperature65 °C   for Duration00:05:00  .

After the Duration00:05:00   at Temperature65 °C  , pause the RT-First Strand program to add the reverse transcription mix.

Remove the plate/microtube from the thermal cycler and place it in a cold rack or TemperatureOn ice   for Duration00:01:00  .

Add Amount9 µL   of the reverse transcription mix to each tube, mix gently, briefly centrifuge, then place in the thermal cycler at Temperature42 °C   for Duration01:30:00  , Temperature85 °C   for Duration00:05:00  , and store at Temperature4 °C  .

1h 35m

Once the RT program has finished, proceed with the next step. Otherwise, store the cDNA at Temperature-20 °C  .

Second Strand Synthesis

Note
The second strand synthesis module is used to generate double-stranded cDNA from first-strand cDNA, as part of the NEBNext non-directional RNA library preparation workflow. 
 
Note 3: Consider processing each cDNA in duplicate.
For the generation of the second strand, mix the following reagents:  
AB
NEBNext® Second Strand Synthesis Reaction Buffer8 µL
NEBNext® Second Strand Synthesis Enzyme Mix4 µL

Dispense Amount12 µL   of the mix per sample into the PCR plate or strip containing the cDNA sample, and mix by pipetting up to 10 times. The final reaction volume will be Amount36 µL  .

Place in the thermal cycler for Duration01:00:00   at Temperature16 °C  , with the lid set to <Temperature40 °C   or turned off.

AMPure Bead Purification

21m 30s

Note
 This step uses beads for the purification of genomic DNA.
Prepare 80% ethanol according to the amount of DNA to be purified.

Vortex the bottle containing the magnetic beads for Duration00:00:30   to ensure they are fully resuspended.

30s

Place the sample DNA into a 1.5 mL LoBind tube.
Note
Note 4: If each sample was processed in duplicate, combine both DNAs into a 1.5 mL LoBind 
microtube.

Add a 1.8X ratio of beads according to the total volume obtained from the second strand synthesis.

Vortex or mix by pipetting at least 10 times.

Incubate for Duration00:05:00   at TemperatureRoom temperature  .

Briefly centrifuge to remove droplets from the walls of the tube.

Place on the magnetic stand until the supernatant becomes clear, approximately Duration00:05:00  .

Remove and discard the supernatant. 
Note
Be careful not to disturb or discard the bead pellet.

Wash with Amount200 µL   of 80% ethanol and incubate for Duration00:00:30   (1/2).
Note
If using 1.5 mL microtubes for purification, perform the washes with Amount300 µL   of ethanol to cover the bead pellet.

30s

Remove and discard the supernatant.

Wash with Amount200 µL   of 80% ethanol and incubate for Duration00:00:30   (2/2).

30s

Remove all ethanol residues with a 10 µL pipette tip.

Briefly centrifuge to concentrate all remaining ethanol at the bottom, place on the magnetic stand for Duration00:01:00  , and remove the ethanol with a 10 µL pipette tip. 
Note
Make sure all ethanol is removed.

Keep the microtube on the magnetic stand with the lid open to dry the beads for up to Duration00:05:00  . 
Note
Avoid over-drying or fragmenting the beads.

Remove the tube from the magnetic stand and resuspend it with Amount35 µL   of nuclease-free water.

Vortex and centrifuge the tube. Verify that the mixture is resuspended.

Incubate for Duration00:02:00   at TemperatureRoom temperature  .

Place on the magnetic stand for Duration00:02:00  .

Transfer Amount30 µL   of the supernatant into a new 1.5 mL LoBind tube.

Amplification

1h 44m

Note
The GenomiPhi V2 DNA Amplification Kit The Illustra™ GenomiPhi™ V2 DNA amplification kit enables whole-genome amplification at a mini scale through isothermal displacement amplification of multiple strands. The amplification is highly uniform across the genome, ensuring the representation is as close as possible to the original DNA sample. The DNA polymerase enzyme used is Phi29, which has proofreading activity. 

Note 5: Consider processing each DNA sample in duplicate.
Add Amount9 µL   of sample buffer in a tube/plate for each sample to be processed.

In a new tube, mix Amount9 µL   of reaction buffer and Amount1 µL   of enzyme for each sample to be processed. 
Mix gently and briefly centrifuge the 'Mix' tube to remove droplets from the lid.
Keep it TemperatureOn ice   or in a cooler.

Add Amount5 µL   of DNA into each well containing the sample buffer and place in the thermal cycler at Temperature95 °C   for Duration00:03:00  .

Then, pause the program and place it TemperatureOn ice   or a cold block for at least Duration00:01:00  . 
After the pause, dispense Amount10 µL   of the amplification mix into each tube of denatured DNA, using a cold block. 
Mix gently.

Place in the thermal cycler and continue the incubation at Temperature30 °C   for Duration01:30:00  , inactivate at Temperature65 °C   for Duration00:10:00  , and then store at Temperature4 °C  .
Note
Note 6: Perform product purification (optional). 

1h 40m

Sequencing

7m 30s

Quantify the samples according to the Qubit DNA High Sensitivity kit procedure and dilute them to Amount0.2 µL  .

In a new 96-well plate, add Amount10 µL   of tagmentation buffer (TB), Amount5 µL   of the amplification mix (ATM), and Amount5 µL   of the PCR product diluted to Amount0.2 µL  . 
Gently mix by pipetting and briefly centrifuge.

Place the 96-well plate in the thermocycler at Temperature55 °C   for Duration00:02:30  , followed by storage temperature at Temperature10 °C  . 
Once the thermocycler reaches Temperature10 °C  , stop the program and proceed to add Amount5 µL   of NTB. 
Mix by pipetting and briefly centrifuge.

2m 30s

Incubate for Duration00:05:00   at TemperatureRoom temperature  . Final volume per well: Amount25 µL  . 
Note
Note 7: Preparing libraries for metagenomic applications requires diluting the indexes to avoid excess adapter dimers when working with low sample concentrations. Index dilution: perform a 1:10 dilution of the indexes, calculating based on the amount of sample you are working with.

For the addition of indexes, add Amount5 µL   of each diluted index (i5 and i7) to the 96-well plate according to the set available in your laboratory. 
Mix by pipetting 5 to 10 times.

Add Amount15 µL   of NPM to each well and mix by pipetting 5 to 10 times. Final volume per well: Amount50 µL  .

Seal the plate with Microseal B or plate strips, briefly centrifuge, and place it in the thermocycler with the following conditions:  
ABC
StepTemperature (°C)Time
Enzyme tagmentation inactivation723 minutes 
Denaturalization and activation9530 seconds 
PCR cycles (16)9510 seconds
5530  seconds 
7230  seconds
Final extension725 minutes
Hold4∞ 
 

At the end of the program, the procedure can be stopped if necessary. The 96-well plate can be stored at Temperature2-8 °C   for up to 2 days; otherwise, proceed with the next step.

Library Purification

12m

Prepare 80% ethanol according to the number of samples to be processed. 
Note
Note 8: The AMPure beads used during library purification should be brought to TemperatureRoom temperature   Duration00:30:00   before use. Homogenize in the tube rotator during this time.

Mix the beads well in the vortex before use and ensure they are homogeneous. Serve a 1:1 ratio of beads/samples in this step.

Cover the plate with adhesive seal B/strip caps and mix using the plate shaker at Shaker1800 rpm, 00:02:00 .

Incubate at TemperatureRoom temperature   for Duration00:05:00  .

Place the plate on the magnetic stand and let it sit for about Duration00:02:00  .

Maintain the plate on the magnetic stand, and carefully remove and discard the supernatant.

With the plate still on the magnetic stand, add Amount200 µL   of 80% ethanol into each well without mixing and incubate for Duration00:00:30   (1/2).

30s

Remove and discard the supernatant.

Add Amount200 µL   of 80% ethanol into each well without mixing and incubate for Duration00:00:30   (2/2).

30s

Remove and discard the supernatant. 
Note
It is important to ensure that most of the supernatant has been removed from the wells.

With the plate still on the magnetic stand, allow air to dry the plate for 10 to 12 minutes at TemperatureRoom temperature  . 
Note
Avoid fragmenting the bead pellet.

Carefully remove the plate from the magnetic stand and add Amount35 µL   of RSB to each well of the plate. Mix up and down 10 times.

Seal the plate and mix at Shaker1800 rpm, 00:02:00 .

Incubate the plate at TemperatureRoom temperature   for about Duration00:02:00  .

Place the plate on the magnetic stand for Duration00:02:00   and allow the beads to form a pellet.

Remove Amount30 µL   of supernatant from the Index PCR Clean-up plate and transfer it to the newly labeled CIA plate. 
Note
Note 9: The protocol can be safely stopped at this point. The CIA plate can be stored in the refrigerator at Temperature2-8 °C  °C or DurationOvernight   at Temperature25 °C   to Temperature15 °C   for 7 days.

Library Quantification

Follow the nucleic acid quantification procedure (Qubit DNA High Sensitivity kit) indicated by the manufacturer for the Qubit fluorometer or the method available in your laboratory for DNA quantification.

Library Pooling

Label a 1.5 mL LoBind microtube as 'PAL' (Pool amplification library).

Add the appropriate number of samples to the previously labeled microtube, as indicated in the 'Pooling library spreadsheet'. The volume of samples will be calculated using the following formula. 

PAL can be brought to a final concentration of Concentration4 nanomolar (nM)  , Concentration2 nanomolar (nM)  , or Concentration1 nanomolar (nM)   in a final volume of approximately Amount25 µL  . The final volume of the PAL and the number of samples can be modified.

Use the resuspension buffer to complete the final volume to which the library will be brought, as indicated in the spreadsheet.

The final concentration of the libraries in the microtube labeled as PAL can be confirmed by quantifying the library again.

Library Purification and Quantification

11m

Prepare Amount3 mL   of 80% ethanol.

Add a 1:1 ratio of AMPure XP beads to the microtube containing the pooled library.

Mix and incubate for Duration00:05:00   at TemperatureRoom temperature  .

Place on the magnetic rack until the supernatant is clear (Duration00:05:00  ).

Remove and discard the supernatant.
Note
 Be careful not to disturb the bead pellet or discard the beads.

Wash the beads with 80% ethanol.

Keep the microtube on the magnetic rack, wash with Amount1000 µL   of 80% ethanol, and incubate for Duration00:00:30   (1/2).

30s

Remove and discard the supernatant.

Wash the beads with Amount1000 µL   of 80% ethanol and incubate for Duration00:00:30   (2/2).

30s

After the second wash, remove all ethanol with a 10 µL pipette tip. 
Then centrifuge to ensure all ethanol is at the bottom, place the tube on the magnetic rack for Duration00:01:00  , and remove with a 10 µL pipette tip.

Remove the tube from the magnetic rack and resuspend it with Amount35 µL   of RSB.

Vortex and centrifuge. Ensure the mixture is resuspended.

Incubate for Duration00:02:00  .

Place on the magnetic rack for Duration00:02:00  .

Transfer Amount30 µL   of the supernatant into a new 1.5 mL microtube labeled 'Purified PAL'.

Quantify the library again to start the dilution and denaturation process. 
Note
Note 10: Depending on the final concentration of your library, proceed to the next step by diluting the sample starting from Concentration4 nanomolar (nM)  , Concentration2 nanomolar (nM)  , or Concentration1 nanomolar (nM)  . If, when quantifying the library, a concentration lower than Concentration1 nanomolar (nM)   is obtained, convert the obtained concentration to picomolar and gradually dilute the library to the indicated loading concentration.

Dilution and Denaturation

Preparation of Fresh NaOH and Dilution

Prepare Concentration1.0 Mass Percent   NaOH.

Dilute Concentration1 Mass Percent   NaOH to Concentration0.2 Mass Percent  . Add the following reagents:  
AB
molecular-grade water800 µL
1.0 N NaOH200 µL

Invert the microtube several times to mix. The result is Amount1 mL   of Concentration0.2 Mass Percent   NaOH.

Denaturation and Dilution of the Library

Combine the following volumes in a 1.5 mL LoBind, depending on your library concentration:  
ABCD
Library concentration1 nM2 nM4 nM
Purified PAL (µL)20105
NaOH 0.2 N (µL)20105
Hyb (µL)960980990
The total volume of the library at 20 pM (µL)1000
 

Mix by vortexing and perform a short centrifugation to concentrate the contents at the bottom of the tube.

Incubate at TemperatureRoom temperature   for Duration00:05:00  .

Add Hyb to the tube containing the denatured library. The result is Amount1 mL   of denatured library at Concentration20 picomolar (pM)  .

Depending on the sequencing chemistry of the kit, perform the corresponding loading dilution. For MiSeq v2, the final loading concentration is up to Concentration10 picomolar (pM)  .

Prepare PhiX at Concentration12.5 picomolar (pM)  . Discard Amount60 µL   of the library and add Amount60 µL   of the PhiX control at Concentration12.5 picomolar (pM)   to achieve a 10% PhiX concentration in the library. 
Mix the solution thoroughly and briefly centrifuge. 

Protocol references

Turbo DNase I, Ambion, Life Technologies. (Pub. No. 1907M REV.J) 
Liz Hughes 2019. RNA precipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.73chqiw  
SuperScript‱ IV First-Strand Synthesis System, Invitrogen‱ Pub. No. MAN0013442 Rev. E.0   
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module, New England Biolabs. Doc. E6111   
Agencourt AMPure XP Beckman Coulter. Doc. B37419AB August 2016   
Illustra GenomiPhi V2 DNA Amplification Kit, Cytiva. 29046315 AC V:3 12/2020

	A	B
	water	2 μL
	SSIV buffer	4 μL
	0.1 M DTT	1 μL
	RNase inhibitor (40 U/μL)	1 μL
	SuperScript IV enzyme	1 μL

	A	B
	NEBNext® Second Strand Synthesis Reaction Buffer	8 µL
	NEBNext® Second Strand Synthesis Enzyme Mix	4 µL

A	B	C
Step	Temperature (°C)	Time
Enzyme tagmentation inactivation	72	3 minutes
Denaturalization and activation	95	30 seconds
PCR cycles (16)	95	10 seconds
	55	30 seconds
	72	30 seconds
Final extension	72	5 minutes
Hold	4	∞

A	B	C	D
Library concentration	1 nM	2 nM	4 nM
Purified PAL (µL)	20	10	5
NaOH 0.2 N (µL)	20	10	5
Hyb (µL)	960	980	990
The total volume of the library at 20 pM (µL)	1000

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Unbiased Metagenomic Protocol: From RNA to Sequencing