Oct 17, 2025
Version 1
One Health metagenomic next-generation sequencing for virus discovery V.1
Forked from a deleted protocol
- Alexandru Tomazatos1,
- Marike Petersen1,
- Balázs Horváth1,
- Alexandra Bialonski1,
- Heike Baum1,
- Marc Lütgehetmann2,
- Alexander Schlaphof1,
- Jaime Costales3,
- M. Alejandra Camacho4,
- Santiago F. Burneo4,
- Petra Emmerich1,
- Ronald von Possel1,
- Daniel Camprubí5,
- Jose Muñoz5,
- Stephanie Jansen6,
- Renke Lühken1,
- Jonas Schmidt-Chanasit1,
- Gábor óth1,
- Dániel Cadar1
- 1Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, 20359 Hamburg, Germany;
- 2University Medical Center Hamburg-Eppendorf, 20359, Hamburg, Germany;
- 3Centro de Investigación Para la Salud en América Latina, Pontificia Universidad Católica del Ecuador, 170525, Quito, Ecuador;
- 4Museo de Zoología, Pontificia Universidad Católica del Ecuador,170525, Quito, Pichincha, , Ecuador;
- 5Barcelona Institute for Global Health (ISGlobal), Hospital Clínic, Universitat de Barcelona, Barcelona, Spain;
- 6Faculty of Mathematics, Informatics and Natural Sciences, Universität Hamburg, 20148 Hamburg, Germany
Protocol Citation: Alexandru Tomazatos, Marike Petersen, Balázs Horváth, Alexandra Bialonski, Heike Baum, Marc Lütgehetmann, Alexander Schlaphof, Jaime Costales, M. Alejandra Camacho, Santiago F. Burneo, Petra Emmerich, Ronald von Possel, Daniel Camprubí, Jose Muñoz, Stephanie Jansen, Renke Lühken, Jonas Schmidt-Chanasit, Gábor óth, Dániel Cadar 2025. One Health metagenomic next-generation sequencing for virus discovery. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5koxnv1b/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 it's working
Created: October 16, 2025
Last Modified: October 17, 2025
Protocol Integer ID: 229977
Keywords: Metagenomics, mNGS, Virus discovery, One Health, Sequencing protocol, Viral enrichment, health pathogen discovery, virus discovery emerging, west nile virus case, emerging viral disease, mosquito homogenate sample, genomic surveillance, free approach for viral detection, accessible tool for outbreak investigation, mosquito homogenate, including dengue, rna virus, sequencing platform, quality genomes for subsequent phylogenomic analysis, dna virus, prior pathogen knowledge, outbreaks of unknown origin, viral detection, most virus, outbreak investigation, viral disease, complete genome recovery, genome recovery, genomic characterization of rna, threat to global health security, traditional diagnostic assay, efficient mngs workflow, quality genome, traditional diagnostic assays such as pcr, microbial nucleic acid, genomic characterization, genome copy, global health security, outbreak
Funders Acknowledgements:
PREPMEDVET
Grant ID: 13N15449
NEED
Grant ID: 01Kl2022
German national program for arbovirus surveillance
Grant ID: 2819107A22
Abstract
Emerging and re-emerging viral diseases pose an increasing threat to global health security. Traditional diagnostic assays such as PCR and serology depend on prior pathogen knowledge and therefore fail to identify novel or divergent agents, especially in outbreaks of unknown origin. Metagenomic next-generation sequencing (mNGS) has emerged as a transformative, hypothesis-free approach for viral detection and genome recovery, yet its implementation remains limited by cost, protocol complexity, and the need for specialized infrastructure. We developed and validated a universal, cost-efficient mNGS workflow optimized for both benchtop and portable Illumina sequencing platforms. The protocol integrates non-specific enzymatic depletion of host and microbial nucleic acids, random priming, and streamlined library preparation using QIAseq FX kit. Analytical sensitivity was evaluated in contrived serum and mosquito homogenate samples spiked with six RNA viruses of public-health importance. Diagnostic performance was further tested on clinical and surveillance specimens, including dengue, chikungunya, monkeypox, COVID-19, and West Nile virus cases. The protocol achieved a detection limit of 10 genome copies µl⁻¹ in human serum and 10² copies µl⁻¹ in mosquito homogenates, with near-complete genome recovery (≥ 90 %) for most viruses at 10³ copies µl⁻¹. In true clinical and field samples, mNGS correctly identified all etiologic agents and generated high-quality genomes for subsequent phylogenomic analysis. Mean sequencing depth across all runs was > 5 × 10⁵ reads per sample on the Illumina iSeq100. This modular mNGS workflow delivers rapid, unbiased detection and genomic characterization of RNA and DNA viruses directly from diverse matrices. Its reduced cost, portability, and shorter turnaround time make it an accessible tool for outbreak investigation, genomic surveillance, and One Health pathogen discovery, particularly in resource-limited environments.
Guidelines
1. Preventing RNA Degradation
- Work quickly and keep samples cold. Always keep RNA and reagents on ice during processing. Minimize freeze–thaw cycles.
- Use RNase-free materials. Use certified RNase-free tubes, pipette tips, and reagents. Autoclaving alone does not inactivate RNases.
- Wear gloves at all times. Skin carries RNases; change gloves frequently and avoid touching surfaces.
- Use RNase inhibitors when applicable. Add RNase inhibitor enzymes to reactions involving sensitive RNA samples.
2. Avoiding RNase Contamination
- Clean the workspace before use. Wipe benches and pipettes with RNase-decontamination solutions (e.g., RNaseZap or 70% ethanol).
- Designate RNA-only areas and equipment. Use separate pipettes and racks for RNA work; label them clearly.
- Use sterile, single-use consumables. Never reuse tubes or tips that have contacted RNA or potential contaminants.
3. Sample Handling and Storage
- Use appropriate buffers. Store RNA in RNase-free water or TE buffer (pH 7.0–8.0).
- Snap-freeze samples in liquid nitrogen and store at −80 °C for long-term preservation. Avoid repeated thawing. Aliquot RNA to minimize freeze–thaw cycles.
4. General Laboratory Practice
- Plan workflow in advance. Prepare reagents, label tubes, and organize materials before beginning.
- Use filtered pipette tips to prevent aerosol contamination.
- Handle reagents gently. Avoid vortexing RNA; mix by pipetting or gentle flicking.
- Document everything. Record RNA concentrations, integrity (e.g., RIN values), and storage conditions.
Materials
Reagents
| A | B | C | D | |
| Reagent | Provider | Cat. no. | Note | |
| QIAamp Viral RNA Mini Kit | Qiagen | 52906 | ||
| Stainless Steel Beads | - | - | For homogenisation | |
| Aluminum Beads | - | - | For homogenisation -80°C | |
| Ultrafree-MC, HV 0,45 µm | Merck Millipore | UFC30HV00 | ||
| Proteinase K | Invitrogen | AM2548 | ||
| Turbo DNAse | Invitrogen | AM2239 | ||
| Baseline Zero DNAse | Epicenter | DB0715K | ||
| Benzonase Nuclease | Merck Millipore | 70664-3 | ||
| RNAse A | Thermo Scientific | EN 0531 | ||
| EDTA 0,5M | Thermo Scientific | AM9260G | ||
| RNAsecure | Invitrogen | AM7006 | ||
| RNAseOUT | Invitrogen | 10777019 | ||
| dNTP Mix | Qiagen | 201901 | ||
| SuperScript IV | Thermo Scientific | 180 90 200 | ||
| Klenow Fragment | NEB | M0212L | ||
| D3N (CAT GCT GAT AGT CGT ACC GCN NNN NNNN) | IDT | - | Primer for RT, used with various concentration! | |
| D3 (CAT GCT GAT AGT CGT ACC GC) | IDT | - | Primer for SISPA | |
| HotStar Taq plus PCR-Kit | Qiagen | 203605 | ||
| QIAseq FX DNA Library UDI Kit | Qiagen | 180477 | with UDI indexes | |
| AMPure XP Beads | Beckman Coulter | A63881 | ||
| Qubit dsDNA HS Assay Kit | Thermo Scientific | Q32854 | ||
| Qubit Assay Tubes | Thermo Scientific | Q32856 | ||
| EB buffer (10 mM Tris-Cl, pH 8.5) | Qiagen | 19086 | ||
| Nuclease free Water | Qiagen | 129114 | ||
| Ethanol | Thermo Scientific | BP28184 | ||
| iSeq 100 i1 Reagent v2 (300-cyc) | Illumina | 20031374 | ||
| Other Illumina reagent for different platforms | Illumina | - |
Necessary reagents for the protocol.
Consumables
| A | B | C | D | |
| Consumable | Provider | Ca. no. | Note | |
| Low binding Tubes 1,5ml | Sarstedt | 72706700 | ||
| Safe Seal SurPhob Tips 10μl | Biozym | VT0200 | ||
| Safe Seal SurPhob Tips 300μl | Biozym | VT0250 | ||
| Biosphere Filter Tips 100μl | Sarstedt | 70.3030.25 | ||
| Biosphere Filter Tips 1250μl | Sarstedt | 701186210 | ||
| Multiply Strip 0,2ml | Sarstedt | 72985002 | ||
| Multichannel reagent reservoirs 25 mL | Integra | 4316 |
Necessary consumables for the protocol.
Equipments
| A | B | C | D | |
| Equipment | Provider | Ca. no. | Note | |
| Eppendorf Research® plus pipette set | Eppendorf | 3123000900 | ||
| DynaMag™-PCR Magnet | Thermo Scientific | 492025 | ||
| BioSan Mini-Zentrifuge/Vortex KombiSpin | PROFILAB BIOSAN | 22004 | ||
| miniPCR® thermal cycler | miniPCR Bio | QP-1016-01 | ||
| Qubit 4 Fluorometer | Thermo Scientific | Q33238 | ||
| PCR Workstation | StarLabs | E2396-9693 | ||
| iSeq100 Instrument | Illumina | 20021532 | ||
| Other Illumina platform | Illumina | - |
Necessary equipments for the protocol.
Safety warnings
!CAUTION.
Any potentially infectious clinical or environmental samples should be handled and made safe in accordance with biosafety regulations.
!CAUTION.
Please follow local institutional review board guidelines covering the collection and storage of clinical samples for research/diagnostic purposes.
!CAUTION.
RNA is highly susceptible to degradation and contamination; therefore, special care must be taken during all steps of RNA handling, extraction, and downstream processing.
!CAUTION.
To avoid stock contamination master mix preparation and reagents handling should be conducted in a dedicated clean cabinet!
Before start
Read the manufacturer`s instruction and be familiar with the reagents and equipments are involved in this protocol!
Sample preparation
Plasma/serum/CSF/culture supernatants or other cell free fluids
Remove 250 µL of biological material and clarify by centrifugation at 12,000 rpm for 00:05:00 . (if available volume is less than 250 µL , use 1xPBS to bring to 250 µL )
Transfer the debris free supernatant on a Ultrafree-MC HV 0.45μm sterile filter (Millipore).
Centrifuge at 8,000 rpm for 00:05:00 , then transfer 200 µL filtrate to a 1.5 ml tube. Proceed to step 5 (Viral Enrichment).
Feces sample
Take ~ 200 mg fecal sample and add 800 µL 1x PBS (Phosphate-Buffered Saline).
Vortex vigorously for ~00:05:00 until the fecal mass is fully homogenized.
Note
Homogenisation with beads is optional.
Clarify the supernatant by centrifugation at 12,000 rpm for 00:05:00 .
Remove 300 µL supernatant into fresh tube. Keep original tube at -80 °C for future extractions.
Clarify the new tube by centrifugation at 12,000 rpm for 00:05:00 .
Transfer 250 µL the debris free supernatant on a Ultrafree-MC HV 0.45μm sterile filter (Millipore).
Centrifuge at 8,000 rpm for 00:05:00 , then transfer 200 µL filtrate to a 1.5 ml tube. Proceed to step 5 (Viral Enrichment).
Tick sample
Place 1 tick into 2ml Tube.
Put the Tube in liquid nitrogen and incubate for 00:05:00
Add stainless steel beads to the tube and homogenize at 30hz for 00:03:00
Add 500 µL cooled (4 °C ) 1x PBS and homogenize at 30hz for 00:03:00 .
Centrifuge the sample at 8,000 rpm for 00:02:00 .
Transfer 250 µL homogenate supernatant to a new 1,5ml Tube and add 2.5 µL Proteinase K. Incubate at 50 °C for 00:30:00 .
Centrifuge at 12,000 rpm for 00:05:00 .
Transfer 250 µL the debris free supernatant on a Ultrafree-MC HV 0.45μm sterile filter (Millipore).
Centrifuge at 8,000 rpm for 00:05:00 , then transfer 200 µL filtrate to a 1.5 ml tube. Proceed to step 5 (Viral Enrichment).
Tissue samples
Take ~ 200 mg tissue sample and add 500 µL 1x PBS (Phosphate-Buffered Saline).
Homogenize at 30hz for 00:03:00 .
Centrifuge the sample at 8000rpm for 00:02:00 .
Transfer 250 µL homogenate supernatant to a new 1,5ml Tube and add 2.5 µL Proteinase K. Incubate at 50 °C for 00:30:00 .
Centrifuge at 12,000 rpm for 00:05:00 .
Transfer 250 µL the debris free supernatant on a Ultrafree-MC HV 0.45μm sterile filter (Millipore).
Centrifuge at 8,000 rpm for 00:05:00 , then transfer 200 µL filtrate to a 1.5 ml tube. Proceed to step 5 (Viral Enrichment).
Viral Enrichment
The nuclease treatment should be prepared on ice/coolrack. Prepare the following Nuclease Mix:
Reagent Amount
Turbobuffer 10x 24 µL
Turbo DNAse 7 µL
Baseline Zero DNAse 3 µL
Benzonase 3 µL
RNAse A 2 µL
Final volume: 39 µL
Add to the 200 µL sample and incubate at 37 °C for 01:30:00 .
Inactivate the nucleases by adding 25 µL of 30 millimolar (mM) EDTA and 10.5 µL RNAsecure to each sample and incubate for 00:10:00 at 65 °C in a heating block.
RNA extraction
Extract RNA from 140 µL sample matrix using the QIAamp Viral RNA Mini Kit according to the manufacturer’s instructions without adding the carrier RNA, elution should be conducted with 60 µL of elution buffer (EB).
Note
The manual for QIAamp Viral RNA Mini Kit:
HB-0354-008_HB_QA_Viral_RNA_Mini_0623_WW.pdf After elution add 1 µL l RNase OUT to the extracted RNA and mix it gently.
Reverse Transcription
Prepare everything on ice/coolrack. Add 1 µL D3N 100µM primer to each well/tube then add 10 µL freshly extracted RNA and mix carefully.
Note
The sequence of D3N primer: 5`-CAT GCT GAT AGT CGT ACC GCN NNN NNNN-3`
Incubate in a PCR machine for 00:10:00 at 75 °C .
Prepare the following reaction mix on ice/coolrack:
Component Amount
5xSSIV Buffer 4 µL
dNTP Mix 1.5 µL
Superscript IV RT 1 µL
RNAse OUT, 40 U/µl 1 µL
Final volume: 8.5 µL
Add 8.5 µL to each reaction and mix by pipetting. Incubate with the following conditions.
Temperature Time
25 °C 00:05:00
50 °C 01:00:00
70 °C 00:15:00
4 °C Till further processes
Second strand synthesis
Prepare the following master mix on ice/coolingrack.
Component Amount
D3N 20µM 1 µL
NE Buffer 2 10x 2.5 µL
dNTP-Mix 10µM 1 µL
Final volume: 4.5 µL
Add 4.5 µL master mix to each tube and mix it by pipetting. Incubate the tubes with the following conditions.
Temperature Time
95 °C 00:02:00
4 °C 00:05:00
Add 1 µL Klenow-Fragment (5000U/ml, NEB) to each reaction and mix it by pipetting. Incubate the tubes with the following conditions.
Temperature Time
37 °C 01:00:00
75 °C 00:20:00
4 °C Till further processes
Clean-up with AMPure XP Beads
Sample/Bead ratio = 1:1.8 (25 µL sample/45 µL bead)
Washing: 3 times with 200 µL 80% EtOH
Elution: 25 µL elution buffer (EB)
Note
Before usage, beads should be at room temperature and mix properly.
Add 45 µL AMPure XP beads to the sample and mix gently.
Incubate for 00:05:00 at room temperature.
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.
Remove supernatant and discard.
Repeat washing step with 80% ethanol additional two times (Step 22-23).
Carefully remove the supernatant and dry the beads approximately for 00:05:00 by leaving the lid open (Dull pellet indicates dryness).
Remove tube from the magnetic rack and resuspend the pellets in 25 µL elution buffer (EB) and incubate at room temperature for 00:02:00
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer 23 µL eluate into clean tube. Make sure that no beads are transferred alongside the supernatant.
Note
5 µL of this dsDNA is used for the next reaction, the rest is stored at -20 °C .
PCR Amplification (SISPA)
Prepare the following master mix on ice/coolingrack.
Component Amount
Buffer 10x 5 µL
dNTP Mix 1 µL
MgCl2 4 µL
Primer D3, 100µM 1 µL
Hot Start Taq Polymerase 0.5 µL
Nuclease free water 33.5 µL
Final volume: 45 µL
Add 45 µL PCR Mix to a new PCR Tube or 8 Strip.
Add 5 µL dsDNA (Sample) to each reaction and mix well by pipetting. Incubate the reaction with the following PCR conditions
Temperature Time Cycles
95 °C 00:15:00 1
94 °C 00:00:30 30
55 °C 00:00:45 30
72 °C 00:01:30 30
72 °C 00:10:00 1
4 °C Till further processes
Spin down the tubes briefly.
Clean-up with AMPure XP Beads
Sample/Bead ratio = 1:0.9 (50 µL sample/45 µL bead)
Washing: 3 times with 200 µL 80% EtOH
Elution: 25 µL elution buffer (EB)
Note
Before usage, beads should be at room temperature and mix properly.
Add 45 µL AMPure XP beads to the sample and mix gently.
Incubate for 00:05:00 at room temperature.
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.
Remove supernatant and discard.
Repeat washing step with 80% ethanol additional two times (Step 22-23).
Carefully remove the supernatant and dry the beads approximately for 00:05:00 by leaving the lid open (Dull pellet indicates dryness).
Remove tube from the magnetic rack and resuspend the pellets in 25 µL elution buffer (EB) and incubate at room temperature for 00:02:00
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer 23 µL eluate into clean tube. Make sure that no beads are transferred alongside the supernatant.
Quantification with Qubit Fluorometer
Quantify 1 µL cleaned PCR product with the Qubit 1x dsDNA HS Assay kit.
Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.
Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1-2). The final volume is 200 µL .
Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid.
Incubate at room temperature for 00:02:00 before measuring.
Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
Note
The manual for Qubit 1x dsDNA HS Assay kit. 
Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf
Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf Note
Alternative dsDNA quantification systems can be used as well (e.g.QuantiFluor ONE dsDNA System).
Normalisation and dilution
The NGS library preparation is conducted with the QIAseq FX DNA Library Kit from Qiagen. The input amount should be 100 ng in 35 µL . Dilute the sample with nuclease free water to reach the suitable concentration.
Component Amount
100 ng amplicon x µL
Nuclease Free water 35-x µL
Note
The leftover from amplicons could be stored at -20 °C .
NGS Library Preparation
Fragmentation and end repair
Note
The manual for QIAseq FX DNA Library Kit. HB_QIAseq_FX_DNA_Library_0624_WW.pdf
HB_QIAseq_FX_DNA_Library_0624_WW.pdf Prepare the following master mix on ice/coolingrack.
Component Amount
FX Buffer 10x 5 µL
FX Enzym Mix 10 µL
Final volume: 15 µL
Add 15 µL master mix to 35 µL (previously diluted to 100 ng /35 µL ) sample and mix it with pipetting. Final volume is 50 µL .
Incubate with the following thermal profile:
Temperature Time
4 °C 00:01:00
32 °C 00:08:00
65 °C 00:30:00
4 °C Till further processes
Adapter Ligation
Note
Important!
-Track the barcodes from each adapter well used for each sample.
-Only 1 adapter should be used per ligation reaction.
-If adapters are used from another supplier, follow the manufacturer`s instructions.
-Do not use adapter wells, if the foil seal has been pierced.
Prepare the following master mix on ice/coolingrack.
Component Amount
DNA Ligase Buffer 5x 20 µL
DNA Ligase 10 µL
Nuclease free water 15 µL
Final volume: 45 µL
Add 5 µL UDI indexes to each reaction. The master mix volume is 50 µL .
Note
UDI indexes
-Unique dual indexing is a sequencing strategy that has distinct, unrelated index sequences for each of the i5 and i7 index reads.
-Unique dual indexing is a known mitigation for filtering index-hopped reads seen in downstream analyses. Misassigned reads will be flagged as "undetermined reads" and can be excluded from analysis.
index-hopping-white-paper.pdf Add the whole ligation master mix (50 µL ) to the whole amount of sample (50 µL ) and mix it via pipetting. The total volume is 100 µL .
Incubate the samples at 20 °C for 00:15:00 .
Clean-up with AMPure XP Beads
Sample/Bead ratio = 1:0.8 (100 µL sample/80 µL bead)
Washing: 2 times with 200 µL 80% EtOH
Elution: 52.5 µL elution buffer (EB)
Note
Before usage, beads should be at room temperature and mix properly.
Add 45 µL AMPure XP beads to the sample and mix gently.
Incubate for 00:05:00 at room temperature.
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.
Remove supernatant and discard.
Repeat washing step with 80% ethanol (Step 22-23).
Carefully remove the supernatant and dry the beads approximately for 00:05:00 by leaving the lid open (Dull pellet indicates dryness).
Remove tube from the magnetic rack and resuspend the pellets in 52.5 µL elution buffer (EB) and incubate at room temperature for 00:02:00
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer 50 µL eluate into clean tube. Make sure that no beads are transferred alongside the supernatant.
Clean-up with AMPure XP Beads
Sample/Bead ratio = 1:1 (50 µL sample/50 µL bead)
Washing: 2 times with 200 µL 80% EtOH
Elution: 26 µL elution buffer (EB)
Note
Before usage, beads should be at room temperature and mix properly.
Add 50 µL AMPure XP beads to the sample and mix gently.
Incubate for 00:05:00 at room temperature.
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.
Remove supernatant and discard.
Repeat washing step with 80% ethanol (Step 22-23).
Carefully remove the supernatant and dry the beads approximately for 00:05:00 by leaving the lid open (Dull pellet indicates dryness).
Remove tube from the magnetic rack and resuspend the pellets in 26 µL elution buffer (EB) and incubate at room temperature for 00:02:00
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer 23.5 µL eluate into a clean PCR tube. Make sure that no beads are transferred alongside the supernatant.
Library amplification
Prepare the following master mix on ice/coolingrack.
Component Amount
HIFI PCR Master Mix 2x 25 µL
Universal Primer Mix 1.5 µL
Final volume: 26.5 µL
Add 26.5 µL master mix to 23.5 µL sample. The total volume is 50 µL . Incubate with the following cycling conditions.
Temperature Time Cycles
98 °C 00:02:00 1
98 °C 00:00:20 8
60 °C 00:00:30 8
72 °C 00:00:30 8
72 °C 00:01:00 1
4 °C Till further processes
Clean-up with AMPure XP Beads
Sample/Bead ratio = 1:1 (50 µL sample/50 µL bead)
Washing: 2 times with 200 µL 80% EtOH
Elution: 52.5 µL elution buffer (EB)
Note
Before usage, beads should be at room temperature and mix properly.
Add 50 µL AMPure XP beads to the sample and mix gently.
Incubate for 00:05:00 at room temperature.
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.
Remove supernatant and discard.
Repeat washing step with 80% ethanol (Step 22-23).
Carefully remove the supernatant and dry the beads approximately for 00:05:00 by leaving the lid open (Dull pellet indicates dryness).
Remove tube from the magnetic rack and resuspend the pellets in 52.5 µL elution buffer (EB) and incubate at room temperature for 00:02:00
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer 50 µL eluate into clean tube. Make sure that no beads are transferred alongside the supernatant.
Library quantification with Qubit and Agarose Gel
Gel Electrophoresis
Running the Gel is optional. If the parameters in this protocol have been followed as stated the samples should range around 450bp and you can use this for the further calculations.
Load 5 µL librariy on a 2% Agarose Gel and let it run at 100 Volts for 45-60min.
The library should show a distribution centered around the size of the fragmented DNA plus 120 bp (450 bp). The increase in library length reflects the addition of sequencing adapters to the DNA fragments or not sufficient fragmentation.
Qubit Measurment
Quantify 1 µL cleaned Final Library with the Qubit 1x dsDNA HS Assay kit.
Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.
Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1-2). The final volume is 200 µL .
Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid.
Incubate at room temperature for 00:02:00 before measuring.
Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
Note
The manual for Qubit 1x dsDNA HS Assay kit. Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf
Qubit_1X_dsDNA_HS_Assay_Kit_UG.pdf Note
Alternative dsDNA quantification systems can be used as well (e.g.QuantiFluor ONE dsDNA System).
Preparing the sequencing run (iSeq)
Thaw the cartridges according to the manufacturer`s instruction.
Summary of Thaw Methods for iSeq cartridge
The samples are entered directly on the device or a sample sheet in CSV format. If you are using sample sheet prepare it in advance.
Note
Using the QIAseq FX DNA Library Prep with UDIs Template for the Local Run Manager (LRM) v2 Excel File from Illumina
PROM-16094-002_TF_QIAseqFX_UDI_LRM_0922_WW.xlsx Mix the samples in equimolar concentration. Mix it properly!
Dilute the mixed libraries in 100 µL volume using EB to 1 nanomolar (nM) concentration.
From previous dilution, make a dilution in 100 µL volume using EB to 50 picomolar (pM) concentration. This means take 95 µL EB and 5 µL diluted library.
Dilute your PhiX Control to 50 picomolar (pM) . The concentration of the Original Tube is 10 nanomolar (nM) .
To your final 100µl of 50pM library add 2µl of the 50pM PhiX. That equals to a spike in of ~2%.
Keep going your library is ready to load! :)
Start the sequencing run (iSeq)
On the iSeq open the “Illumina iSeq Control Software”.
Press the “Sequence” button on the center of the screen. The device will open and you can watch a video explaining the next steps.
Remove the thawed cartridge and the room temperature flow cell from the foil bags.
Invert the cartridge 5 times and lightly tap it on your work surface to help the reagents settle to the bottom.
Pierce the silver foil on the cartridge with a pipette tip.
With a new pipette tip load 20 µL of the finished 50 picomolar (pM) pool to the bottom of the cartridge.
Insert the flowcell into the cartridge.
Place the Cartridge into the iSeq and select “close door”.
In the “Run Setup” your run should be automatically selected (if you already added the sample sheet).
Once the device has checked the cartridge and flow cell and you have double checked the details you can press “start run”.
Bioinformatic analysis
The generated FASTQ files are processed using MegaVir.
MegaVir is a bioinformatic pipeline for virome characterization and genome assembly.
The workflow of the MegaVir pipeline.
Note
The link for the pipeline:
Acknowledgements
This research was financed by the German Federal Ministry of Education and Research within project PREPMEDVET no. 13N15449. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript/protocol.
RL is funded by the Federal Ministry of Education and Research of Germany (BMBF) under the project NEED (01Kl2022). Mosquitoes and dead birds were collected as part of the German national program for arbovirus surveillance funded by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE) with the grant number 2819107A22.