Jan 31, 2023
Version 1
Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics V.1
- Lily Tong1,
- Katherine Smollett1,
- Kirsty Kwok1,
- Kyriaki Nomikou1,
- Ma. Jowina Galarion1,
- Daniel Mair1,
- Ana Filipe1,
- Jenna Nichols2
- 1MRC-University of Glasgow Centre for Virus Research;
- 2University of Glasgow
- CVR Genomics
Protocol Citation: Lily Tong, Katherine Smollett, Kirsty Kwok, Kyriaki Nomikou, Ma. Jowina Galarion, Daniel Mair, Ana Filipe, Jenna Nichols 2023. Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge34zol47/v1
Manuscript citation:
Antonia Ho, Richard Orton, Rachel Tayler, Patawee Asamaphan, Vanessa Herder, Chris Davis, Lily Tong, Katherine Smollett, Maria Manali, Jay Allan, Konrad Rawlik, Sarah E. McDonald, Elen Vink, Louisa Pollock, Louise Gannon, Clair Evans, Jim McMenamin, Kirsty Roy, Kimberly Marsh, Titus Divala, Matthew TG Holden, Michael Lockhart, David Yirrell, Sandra Currie, Maureen O’Leary, David Henderson, Samantha J. Shepherd, Celia Jackson, Rory Gunson, Alasdair MacLean, Neil McInnes, Amanda Bradley-Stewart, Richard Battle, Jill Hollenbach, Paul Henderson, Miranda Odam, Primrose Chikowore, Wilna Oosthuyzen, Meera Chand, Melissa Shea Hamilton, Diego Estrada-Rivadeneyra, Michael Levin, Nikos Avramidis, Erola Pairo-Castineira, Veronique Vitart, Pablo Murcia, Craig Wilkie, Surajit Ray, DIAMONDS consortium, ISARIC4C Investigators, Massimo Palmarini, David L. Robertson, Ana Filipe, Brian J. Willett, Judith Breuer, Malcolm G. Semple, David Turner, J Kenneth Baillie, Emma C. Thomson (in press). Adeno-associated virus 2 infection in children with non-A-E hepatitis. Nature.
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 14, 2022
Last Modified: January 31, 2023
Protocol Integer ID: 71354
Keywords: Viral genomics, Next-generation sequencing, viral discovery, Illumina, Metagenomics, RNA, DNA, Untargeted detection of viruses, powerful tool for viral genomic, virus specific primer, viral genomic, rna viral read, metagenomic sequencing, detection of viral nucleic acid, prior knowledge of the virus, based targeted enrichment, viral nucleic acid, sequencing library, methods for specific enrichment, viral read, detecting rna, metagenomic, metagenomic library, protocol discovery of rna, acute hepatitis of unknown aetiology, targeted enrichment, dna virus, generation sequencing, discovery of rna, simultaneous extraction of rna, unknown virus, acute hepatitis, associated virus, enrichment such as pcr amplification, rna, deplete host nucleic acid, approach to an outbreak, specific enrichment, detection of dna, virus, enrichment, identification of adenovirus, unwanted nucleic acid, human herpesvirus, dna sample, adenovirus, outbreak, nucleic acid, depletion of unwanted nucleic acid,
Abstract
Next-generation sequencing is a powerful tool for viral genomics. Viruses often constitute a very small proportion of any given sample meaning that methods that enable detection of viral nucleic acids are frequently needed for detection and characterisation. Improvement of sensitivity can be achieved by depletion of unwanted nucleic acid during sample pre-treatment or by enrichment such as PCR amplification with virus specific primers, or probe-based targeted enrichment. However, some methods for specific enrichment rely on prior knowledge of the viruses. Here we describe a method for next-generation sequencing to identify unknown viruses (see Figure 1).
Figure 1: Diagram of workflow for discovery of DNA and RNA viruses using metagenomic sequencing. Optional ribosomal depletion step is shown in hashed box. Image prepared using BioRender.com.
Following simultaneous extraction of RNA and DNA samples are split into two and subjected to non-specific treatments that deplete host nucleic acids and improve chances of detecting RNA or DNA viruses, respectively. Illumina sequencing libraries are then prepared and these metagenomic libraries can directly sequenced. These sequencing libraries can then be subjected to targeted enrichment using a pan-viral probe set to achieve higher sensitivity (for details see accompanying protocol Discovery of RNA and DNA viruses using next-generation sequencing: Targeted enrichment).
We applied this approach to an outbreak of acute hepatitis of unknown aetiology in children, enabling the identification of adeno-associated virus 2 (AAV2) in all patients but not in samples from controls. This method also led to the identification of adenovirus and human herpesviruses.
This protocol describes the pre-treatment of nucleic acid extracts to improve detection of DNA and RNA viral reads followed by Illumina library preparation and metagenomic sequencing.
Materials
Reagents:
Ambion™ DNase I (RNase-free)Thermo FisherCatalog #AM2224
Agencourt RNAClean XP Magnetic BeadsBeckman CoulterCatalog #A63987
Illumina Ribo-Zero Plus rRNA Depletion KitIllumina, Inc.Catalog #20040526
Deoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
Random hexamersThermo ScientificCatalog #N8080127
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080044
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - 100 rxnsNew England BiolabsCatalog #E6111L
Agencourt AmPure XP beadsCatalog #A63880
NEBNext Microbiome DNA Enrichment Kit - 24 rxnsNew England BiolabsCatalog #E2612L
KAPA LTP library prep kitRocheCatalog #796188001
40% Polyethylene Glycol MW 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1458
5 M Sodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5150-1L
NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)New England BiolabsCatalog #E6440-8
HotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851 Qubit RNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32852
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Genomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366
Additional reagents required:
Absolute ethanol
Nuclease-free water
10 mM Tris pH8
Equipment:
Equipment
LE220
NAME
High-throughput focused ultrasonicators
TYPE
Covaris
BRAND
500569
SKU
LINK
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Protocol materials
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Illumina Ribo-Zero Plus rRNA Depletion KitIllumina, Inc.Catalog #20040526
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Agencourt RNAClean XP Magnetic BeadsBeckman CoulterCatalog #A63987
5 M Sodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5150-1L
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851
Agencourt AmPure XP beadsCatalog #A63880
Random hexamersThermo ScientificCatalog #N8080127
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080044
Ambion™ DNase I (RNase-free)Thermo FisherCatalog #AM2224
40% Polyethylene Glycol MW 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1458
HotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
Genomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366
Deoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - 100 rxnsNew England BiolabsCatalog #E6111L
NEBNext Microbiome DNA Enrichment Kit - 24 rxnsNew England BiolabsCatalog #E2612L
KAPA LTP library prep kitRocheCatalog #796188001
NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)New England BiolabsCatalog #E6440-8
Qubit RNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32852
Before start
This protocol starts with nucleic acid extracts.
Initial sample preparation
Quantify the DNA and RNA concentration of your samples using Qubit HS reagents.
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851 Qubit RNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32852
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Note
Depending on the sample type the level of RNA/DNA may be undetectable and so quantification may not be required for all samples.
Split each nucleic acid extract into two subsamples for RNA and DNA virus detection.
If required, make up each sample to 50 µL with Nuclease-free water.
Prepare two 0.2 mL PCR tubes per sample labelled with R (for the RNA pre-treatment) or D (for DNA pre-treatment) along with the sample names.
Note
For multiple samples it is recommended that PCR strip tubes or plates are used, ensure that subsamples are grouped for RNA or DNA virus detection and able to be easily separated.
Add 25 µL sample to the tube R and 25 µL sample to tube D.
Subsample R
Subsample D → proceed to DNA virus detection - Microbiome enrichment (step 14).
Note
It is recommended to process the RNA samples first to minimise degradation.
RNA virus detection - DNase I treatment
27m
Prepare DNase I mix as follows (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 10X DNase I buffer | 3 | |
| DNase I | 2 | |
| Total | 5 |
Ambion™ DNase I (RNase-free)Thermo FisherCatalog #AM2224
Note
For large amounts of DNA use the suppliers recommendations.
Add 5 µL DNase I mix to 25 µL RNA sample .
Incubate as follows:
37 °C for 00:15:00 then place On ice .
15m
Perform clean-up with 2X volume of RNAclean XP magnetic beads.
Agencourt RNAClean XP Magnetic BeadsBeckman CoulterCatalog #A63987
Note
Ensure RNAclean XP beads are equilibrated to room temperature for ~30 min and vortex well before use.
Add 60 µL RNAclean XP beads to the 30 µL DNase I reaction and mix by pipetting.
Incubate at Room temperature for 00:05:00 .
5m
Place on a magnetic rack until beads and solution have fully separated.
Carefully remove supernatant being careful not to disturb the beads.
Wash 2X with 200 µL 80% Ethanol .
Note
Ensure ethanol is freshly prepared.
Remove all traces of ethanol and air dry for up to 00:05:00 .
Note
Take care not to over dry the beads.
5m
To elute DNase I treated RNA add 12 µL Nuclease-free water and incubate at Room temperature for at least 00:02:00
2m
RNA virus detection - Ribosomal depletion
56m
Transfer 10 µL DNase I treated RNA to fresh 0.2 mL tubes/plate for Ribo-zero probe hybridisation.
Illumina Ribo-Zero Plus rRNA Depletion KitIllumina, Inc.Catalog #20040526
Note
Ribo-depletion is recommended for sample that are likely to contain large levels of host or bacterial RNA (such as tissue biopsy, faecal, oral or nasal) but can be excluded from sample with lower levels of host RNA (such as plasma, serum or cerebrospinal fluid), or when the sample input is too low to enable library preparation.
Thaw DB1 and DP1 at Room temperature , vortex to mix and centrifuge briefly.
Prepare the hybridisation probe mix on ice (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| DB1 (Depletion Probe Buffer) | 3 | |
| DP1 (Depletion Probe Pool) | 1 | |
| Total | 4 |
Thoroughly pipette mix.
Add 4 µL hybridisation probe mix to each 10 µL sample and fully mix by pipetting 10 times.
Incubate samples as follows:
95 °C for 00:02:00
Decrease 0.1 °C /sec until temperature reaches 37 °C then hold.
Note
The program takes ∼ 15 min to finish.
15m
Prepare rRNA Depletion.
Thaw RDB and RDE at Room temperature , vortex or flick (RDE) to mix and centrifuge briefly.
Prepare the rRNA Depletion mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| RDB (RNA Depletion Buffer) | 4 | |
| RDE (RNA Depletion Enzyme) | 1 | |
| Total | 5 |
Thoroughly pipette mix.
Add 5 µL rRNA Depletion mix to each 14 µL and fully mix by pipetting 10 times.
Incubate samples as follows:
37 °C for 00:15:00
4 °C hold
15m
Prepare probe removal.
Thaw PRB and PRE at Room temperature , vortex (PRB) or flick (PRE) to mix and centrifuge briefly.
Prepare the Probe Removal mix On ice (for multiple samples prepare a mastermix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| PRB (Probe Removal Buffer) | 7 | |
| PRE (Probe Removal Enzyme) | 3 | |
| Total | 10 |
Thoroughly pipette mix.
Add 10 µL Probe Removal mix to each 19 µL reaction and fully mix by pipetting 10 times.
Incubate samples as follows:
37 °C for 00:15:00
70 °C for 00:15:00
4 °C hold
30m
Perform clean-up with 2X volume of RNAclean XP magnetic beads.
Agencourt RNAClean XP Magnetic BeadsBeckman CoulterCatalog #A63987
Note
Ensure RNAclean XP beads are equilibrated to room temperature for ~30 min and vortex well before use.
Add 60 µL RNAClean XP beads to the 30 µL reaction , mix by pipetting.
Incubate at Room temperature for 00:05:00
5m
Place on a magnetic rack until beads and solution have fully separated.
Remove and discard supernatant.
Wash 2X with 175 µL 80% Ethanol (freshly prepared) .
Remove all traces of ethanol and air dry for up to 00:02:00
Note
Do not over-dry the beads.
2m
Elute in 12 µL ELB (Elution buffer) by incubating at Room temperature for 00:02:00 .
2m
RNA virus detection - Low input reverse transcription
4h
Transfer 10 µL RNA sample to fresh 0.2 mL tubes/plates.
Note
This method for cDNA preparation has been tested for very low-input and undetectable RNA/DNA by Qubit HS reagents.
Prepare NTP/Hex mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 10 mM dNTP | 1 | |
| Random Hexamers | 1 | |
| Total | 2 |
Deoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
Random hexamersThermo ScientificCatalog #N8080127
Add 2 µL NTP/Hex mix to each 10 µL sample .
Incubate as follows:
65 °C for 00:05:00
immediately place On ice
5m
Prepare the SSIII master mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 5X Reverse transcription buffer | 4 | |
| SuperScript III | 2 | |
| RNaseOUT | 1 | |
| DTT | 1 | |
| Total | 8 |
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080044
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Add 8 µL SSIII mix to each 12 µL reaction .
Incubate samples as follows:
25 °C for 00:10:00
55 °C for 01:00:00
70 °C for 00:15:00
4 °C hold
1h 25m
Prepare second strand mix (for multiple samples prepare a mastermix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 10X Second strand synthesis buffer | 8 | |
| Second strand synthesis enzyme | 4 | |
| Nuclease-free water | 48 | |
| Total | 60 |
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - 100 rxnsNew England BiolabsCatalog #E6111L
Add 60 µL second strand mix to each 20 µL reaction
Incubate as follows on PCR machine
16 °C for 02:30:00
4 °C hold
2h 30m
Perform clean up with 1X volume of AmPure XP beads.
Agencourt AmPure XP beadsCatalog #A63880
Note
Ensure AmPure XP beads are equilibrated to room temperature for 30 min and vortex well before use.
Add 80 µL AmPure XP to each 80 µL reaction (1:1 Ampure:sample ratio) and mix well.
Incubate at Room temperature for 00:05:00 .
5m
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200 µL 80% Ethanol (freshly prepared) .
Remove all traces of ethanol and air dry for 00:05:00 .
5m
Elute in 27 µL 10 mM Tris pH8 by incubation at Room temperature for 00:02:00 .
Note
cDNA can be stored at 4°C overnight or -20°C longer term
cDNA → proceed to Section – Library prep (step 22) or store until DNA samples ready so can process together.
2m
DNA virus detection - Microbiome enrichment
4h 43m
Pre-bind MBD2-Fc Protein to Magnetic Beads.
NEBNext Microbiome DNA Enrichment Kit - 24 rxnsNew England BiolabsCatalog #E2612L
See following attachment for reagent calculations:
Microbiome_calculations.xlsx Note
Microbiome enrichment can be used to deplete CpG modified DNA.
Pipette 1 µL Protein A magnetic beads (see column B of reagent calculation table) for every 6.25 ng input DNA into a 1.5 mL DNA LoBind tube.
Note
For low or undetectable amounts of DNA (<30 ng) use 5 μL of magnetic beads.
Note
Do not vortex the magnetic beads.
Add 0.1 V MBD2-Fc protein (see column C of reagent calculation table) to the Protein A magnetic beads.
Mix the bead-protein mixture by placing the tube in a rotating mixer for 00:10:00 .
10m
Prepare the 1X Bind/Wash buffer and keep it On ice :
| A | B | |
| Component | Volume (μl) | |
| 5X NEBNext Bind/Wash buffer | 800 | |
| Nuclease-free water | 3200 | |
| Total | 4000 |
After the incubation, briefly spin the tube and place on the magnetic rack for 00:05:00 until the beads have collected.
5m
Remove the supernatant with a pipette without disturbing the beads.
Add 1 mL 1X Bind/Wash buffer to the tube to wash the beads. Pipet up and down a few times to mix.
Mix the beads on a rotating mixer for 00:03:00 at Room temperature .
3m
Briefly spin the tube and place on the magnetic rack for 00:05:00 until the beads have collected.
5m
Remove the supernatant with a pipette without disturbing the beads.
Repeat wash step (2 washes in total).
Resuspend the beads in the volume of 1X Bind/Wash buffer equal to the initial magnetic bead volume in step 13.1 (see sum of column B of reagent calculation table).
Capture Methylated Host DNA.
Add appropriate volume of 5X Bind/Wash buffer to fresh tubes for each sample to give a 1X solution (see column D of reagent calculation table).
Add the volume of sample to give up to 1 µg DNA (see column A of reagent calculation table).
Note
It is important that the buffer and DNA is combined before adding the beads!
Note
Use a minimum volume of 40 μl and make up with 10 mM Tris pH8 if necessary. Maximum volume is 200 μl.
Add appropriate volume of washed Fc-bead/protein mix (see column B of reagent calculation table) to the DNA/buffer mix.
Mix and incubate in a rotating mixer at RT for 00:15:00 to 04:00:00 depending on sample type .
Note
Although the manufacturer's protocol suggests 15 min we would not recommend less than 30 min, and in some cases much longer is required.
Note
Ensure that the liquid moves freely during the incubation to achieve efficient mixing.
4h 15m
Elute Microbiome DNA.
Briefly centrifuge and place on a magnet for at least 00:05:00 to separate the bead-bound host DNA.
5m
Remove the supernatant containing host depleted/microbiome enriched DNA to fresh tubes.
Note
The supernatant contains the host-depleted microbiome DNA, the beads contain the microbiome-depleted host DNA. If required the beads can be resuspended in 1X bind buffer and stored for analysis of host DNA see manufacturer's guidelines for details.
Note
The supernatant is used directly in the sonication but can be cleaned up with 2X Ampure (see column E of reagent calculation table for volume) for longer term storage.
Agencourt AmPure XP beadsCatalog #A63880
DNA virus detection - sonication
1h 5m
If required make sample up to 55 µL with 10mM Tris pH8 .
Prepare sonicator for use.
Note
Here we describe sonication with the Covaris LE220 and 8microTUBE-50 AFA Fiber Strip V2 or 96 microTUBE AFA Fiber Plate Thin Foil. Be sure to follow the manufacturers recommendations for your own instruments. In addition, some companies offer alternatives to sonication such as the use of fragmentases, this would need to be optimised before use.
Equipment
LE220
NAME
High-throughput focused ultrasonicators
TYPE
Covaris
BRAND
500569
SKU
LINK
Fill the tank with water to FILL level -2.
Switch on the chiller and ensure set to 7 °C .
Switch on the water conditioning system, the Covaris and the computer.
Open the SonoLab software.
Select Home and the transducer will get submerged, the degas pump should start automatically.
Degas the water bath for ~00:45:00 .
45m
Set up the sonication conditions as follows:
| A | B | |
| Peak power | 450 | |
| Duty factor | 10 | |
| Cycles/burst | 1000 | |
| Treatment time (s) | 89 | |
| Dithering | on |
Example sonication conditions to achieve ~350 bp fragments.
Note
The precise conditions will need to be validated using a gDNA control prior to use with samples.
Sonicate samples.
Add 55 µL Sample to either the 8 microTUBE-50 AFA Fiber Strip V2 or 96 microTUBE AFA Fiber Plate Thin Foil.
Place the strip/plate in the appropriate holder and screw into place (ensure that it is set up the same as the program conditions).
Select Load position to move the support arm forward.
Press the green button and open the door to put the holder into the support arm, ensure in the correct orientation and close the door.
Press start position to submerge the rack and confrim the correct volume of water has been added (samples should NOT be fully submerged).
Press Run to start the sonication.
Once completed press Load Position to remove the plate.
Transfer 50 µL sheared DNA to PCR tubes.
Note
Option to check shearing by running 1 μL on TapeStation gDNA.
Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
Genomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
1.4X Ampure clean up.
Agencourt AmPure XP beadsCatalog #A63880
Add 70 µL Ampure XP to the samples (ratio 1.4:1).
Incubate at Room temperature for 00:15:00 .
15m
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200 µL 80% Ethanol (freshly prepared) .
Remove all traces of Ethanol. Air dry for 00:05:00 .
5m
Elute samples in 25 µL 10mM Tris pH8 .
Transfer 25 µL sample to new tubes.
Note
Pause: DNA can be stored at 4°C overnight or -20°C for longer term.
Sheared DNA → proceed to Section – Library prep (step 22) can process alongside the prepared cDNA samples.
Illumina sequencing library preparation
1h 50m
Prepare the End Repair mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 10X End repair buffer | 3 | |
| End repair enzyme | 2 | |
| Total | 5 |
Note
This version uses the KAPA LTP kit that has been discontinued by the supplier. Reactions at at half the volume of the manufacturer's recommendations with the final PCR reaction being at full volume.
Note
Samples pre-treated for the detection of DNA and RNA viruses can be processed alongside each other. Treat as seperate samples and do not pool before library prep.
KAPA LTP library prep kitRocheCatalog #796188001
Add 5 µL End repair mix to each 25 µL sample .
Incubate as follows (if using a PCR machine do not use hot lid):
20 °C for 00:30:00
30m
1.4X Ampure XP clean up.
Agencourt AmPure XP beadsCatalog #A63880
Add 20 µL 10 mM Tris pH8 increase volume of each sample to a total of 50 µL .
Add 70 µL Ampure XP (1.4:1 Ampure:sample ratio). Pipette up and down to mix.
Incubate at Room temperature for 00:05:00 .
5m
Place samples on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200 µL 80% Ethanol (freshly prepared) .
Remove all traces of ethanol and air dry for 00:05:00 .
5m
Elute in 21 µL 10mM Tris pH8 leaving beads in solution.
13. Prepare the A-Tail mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 10X A-Tail buffer | 2.5 | |
| A-Tail enzyme | 1.5 | |
| Total | 4 |
KAPA LTP library prep kitRocheCatalog #796188001
Add 4 µL A-Tail mix to each 21 µL reaction .
Incubate as follows:
30 °C for 01:00:00
1h
1.4X SPRI clean up.
Note
SPRI is a solution containing 20% PEG 8000 and 2.5 M NaCl and allows for Ampure clean up using the beads contained within the reaction. SPRI is included as art of the KAPA LTP library prep kit but if required additional SPRI can be made by mixing equal quantities of 40% PEG 8000 and 5 M NaCl.
40% Polyethylene Glycol MW 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1458
5 M Sodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5150-1L
Add 25 µL 10 mM Tris pH8 to increase volume of each sample to a total of 50 µL .
Add 70 µL SPRI (1.4:1 SPRI:sample ratio). Pipette up and down to mix.
Note
The magnetic beads should remain in solution form the previous step, alternatively Ampure XP can be used instead of SPRI reagent.
Incubate at Room temperature for 00:05:00 .
5m
Place the samples on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200 µL 80% Ethanol (freshly prepred) .
Remove all traces of ethanol and air dry for 00:05:00 .
5m
Elute in 15 µL 10 mM Tris pH8 leaving beads in solution.
Quantify 1 µL using Qubit high sensitivity dsDNA.
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Calculate the amount of pmol per in each 14 µL A-tail reaction as follows (alternatively use the calculation in the attached sheet):
Note
If the fragment size is not known estimate it to be 350 bp.
LTP_adapterCalcultion.xlsx Calculate the amount of adapter required (aim for 20:1 ratio adapter to sample - see calculation sheet).
Note
The adapter is contained in the NEBNext multiplex oligo kit.
NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)New England BiolabsCatalog #E6440-8
Dilute adapter in water to achieve appropriate concentration in a total volume of 5 µL per reaction.
Note
If DNA is undetectable by Qubit HS reagents use 5 μL of 0.15 μM adapter.
Prepare the Ligation mix (for multiple samples prepare a master mix with 10% excess):
| A | B | |
| Component | Volume (μl) | |
| 5X ligation buffer | 5 | |
| DNA ligase | 2.5 | |
| Total | 7.5 |
KAPA LTP library prep kitRocheCatalog #796188001
Add 7.5 µL Ligation mix to 14 µL reaction (ensure remains on the beads).
Add 5 µL diluted NEBNext adapter .
Incubate as follows (if using a PCR machine ensure the hot lid is not turned on):
20 °C for 01:00:00 .
1h
Add 0.75 µL USER enzyme to each tube.
Note
USER enzyme is included as part of the NEBNext multiplex oligo kit.
NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)New England BiolabsCatalog #E6440-8
Step 15 Incubate as follows:
37 °C for 00:15:00
4 °C hold
15m
0.9X SPRI clean up.
Add 25 µL 10 mM Tris pH8 to make volume up to 50 µL .
Add 45 µL SPRI to the samples (ratio 0.9:1).
Incubate at Room temperature for 00:05:00 .
5m
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200 µL 80% Ethanol (freshly prepared) .
Remove all traces of ethanol and air dry for up to 00:05:00 .
Note
Take care not to over-dry the beads.
5m
Elute samples in 22 µL 10 mM Tris pH8 .
Transfer 20 µL to new freah 0.2 mL PCR tubes.
Add 5 µL NEBNext multiplex oligos each sample.
Note
It is essential the UDI primers are used to prevent index hopping on the sequencer. It is recommended that the primer kits are rotated between batches, there are currently 5 NEBNext multiplex oligo kits.
NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)New England BiolabsCatalog #E6440-8
Add 25 µL KAPA HotStart Ready mix to each sample.
Note
KAPA HotStart Ready mix is contained within the KAPA LTP kit but may need to be topped up with additional reagent.
KAPA LTP library prep kitRocheCatalog #796188001 HotStart ReadyMix (KAPA HiFi PCR kit)Kapa BiosystemsCatalog #KK2601
Incubate as follows:
98 °C for 00:00:45
4-20* cycles of
98 °C for 00:00:15
65 °C ** for 00:00:30
72 °C for 00:00:30
Final cycle of
72 °C for 00:01:00
4 °C hold 4oC hold
Note
*The number of cycles depends on the input DNA concentration, too few cycles and not enough library is produced, too many results in a high proportion of PCR duplicates and daisy chains.
| A | B | C | |
| Input DNA (into ER and AT) | Number of cycles required to generate: | ||
| 100 ng library | 1 μg library | ||
| 1 μg | 3 | 3 | |
| 500 ng | 3 | 3–4 | |
| 250 ng | 3 | 4–6 | |
| 100 ng | 3 | 6–7 | |
| 50 ng | 3–5 | 7–8 | |
| 25 ng | 5–7 | 8 – 10 | |
| 10 ng | 7–9 | 11 – 13 | |
| 5 ng | 9 – 11 | 13 – 14 | |
| 2.5 ng | 11 – 13 | 14 – 16 | |
| 1 ng | 13 – 15 | 17 – 19 | |
KAPA recommendations for number of PCR cycles required for different amounts of input DNA. As the PCR primers contain the full Illumina adapter/index sequences at least 3 cycles are required.
Note
**The annealing temperature of 65°C is optimal for the NEBNext primers, if other primers are used this may need to be altered.
Note
It is recommended that after this step (i.e. post-PCR when samples are more concentrated and contain common adapters) work continues in a different area to prevent contamination.
3m
Clean up and QC the libraries as in protocol Library clean up and quality control for Illumina sequencing.
Metagenomic sequencing
Metagenomic sequencing can speed up identification of any viruses in the samples and, depending on the virus and viral load, may be suficient to generate full genomes.
Using the bp size and ng/μl concentration calculate the nM concentration for each library as follows:
Pool the libraries by equal molarity and QC the pools as described in the protocol Library pooling and quality control for Illumina sequencing.
Note
Ensure that enough unpooled library remains to perform targeted enrichment .
Sequence the pools on an Illumina sequencer following the manufacturer's guidelines.
Note
For metagenomic viral discovery sequencing we recommend sequencing at 40 million reads per sample (20 million for the RNA viral detection and 20 million for the DNA viral detection).