Feb 14, 2020
Version 3
Sequence-Independent, Single-Primer Amplification of RNA viruses V.3
- 1University of Wisconsin - Madison
Protocol Citation: Gage Moreno, David O'connor 2020. Sequence-Independent, Single-Primer Amplification of RNA viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.bckxiuxn
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: February 14, 2020
Last Modified: February 14, 2020
Protocol Integer ID: 33143
Abstract
This protocol outlines the methods to perform unbiased direct metagenomic sequencing of nucleic acid extracts from cell-free fluids. This protocol can be adapted to be run on Illumina and Nanopore sequencing platforms.
The protocol is based off of the work from Kafetzopoulou et al. (PMID: 30563591). Liana has provided the lab with detailed protocols, and has worked with us extensively on optimizing and getting protocols running efficiently. Please note that this protocol has been updated to use SuperScript IV with it’s optimal temperature which has been reflected in Lewandowski et al. (DOI: https://doi.org/10.1128/JCM.00963-19)
Notes:
* This protocol has been used to sequence influenza direction from respiratory clinical samples (DOI: https://doi.org/10.1128/JCM.00963-19).
* A team from China published last week on a 2019-nCoV familial cluster using the SISPA protocol as for coronavirus whole genome sequencing (DOI: https://doi.org/10.1016/S0140-6736(20)30154-9)
SISPA-Primer A - 5'-GTT TCC CAC TGG AGG ATA-(N9)-3'
SISPA-Primer B - 5′-GTT TCC CAC TGG AGG ATA-3′
Materials
MATERIALS
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Ampure XP beads Beckman CoulterCatalog #A63881
QIAamp® Viral RNA Mini QiagenCatalog #52906
RNase HNew England BiolabsCatalog #M0297S
Linear acrylamideThermofisherCatalog #AM9520
Filter (0.22µm)CostarCatalog #8110
SuperScript™ IV First-Strand Synthesis SystemThermo FisherCatalog #18091200
Sequenase Version 2.0 DNA PolymeraseThermo FisherCatalog #70775Y200UN
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
dNTP Mix (10 mM each)Thermo FisherCatalog #R0192
AccuTaq LA DNA PolymeraseMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8045-125UN
SISPA-Primer AIDT
SISPA-Primer BIDT
STEP MATERIALS
Filter (0.22µm)CostarCatalog #8110
Linear Acrylamide (5 mg/ml) (1 ml Tube)Thermo FisherCatalog #AM9520
QIAamp® Viral RNA Mini QiagenCatalog #52906
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Protocol materials
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
RNA Clean & Concentrator™-5Zymo ResearchCatalog #R1015
Ampure XP beads Beckman CoulterCatalog #A63881
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
RNase HNew England BiolabsCatalog #M0297S
Linear acrylamideThermofisherCatalog #AM9520
Filter (0.22µm)CostarCatalog #8110
SuperScript™ IV First-Strand Synthesis SystemThermo FisherCatalog #18091200
SISPA-Primer BIDT
Filter (0.22µm)CostarCatalog #8110
Linear Acrylamide (5 mg/ml) (1 ml Tube)Thermo FisherCatalog #AM9520
QIAamp® Viral RNA Mini QiagenCatalog #52906
QIAamp® Viral RNA Mini QiagenCatalog #52906
Sequenase Version 2.0 DNA PolymeraseThermo FisherCatalog #70775Y200UN
AccuTaq LA DNA PolymeraseMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8045-125UN
SISPA-Primer AIDT
dNTP Mix (10 mM each)Thermo FisherCatalog #R0192
Filter (0.22µm)CostarCatalog #8110
Linear Acrylamide (5 mg/ml) (1 ml Tube)Thermo FisherCatalog #AM9520
QIAamp® Viral RNA Mini QiagenCatalog #52906
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Nucleic Acid Extraction
Nucleic Acid Extraction
Add 280µL cell-free liquid to 0.22µm centrifuge filter.
Note: (add in 2x what you want to get out)
Filter (0.22µm)CostarCatalog #8110
Centrigue at 14,000 RPM for 5 minutes
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
14000 rpm, 00:05:00
Transfer 140µL of filtered sample to a new tube.
Prepare Buffer AVL and linear polyacrylamide mix.
* For one sample, 560µL Buffer AVL + 5.6µL linear polyacrylamide Reagent Volume (µL)
Linear Acrylamide (5 mg/ml) (1 ml Tube)Thermo FisherCatalog #AM9520
QIAamp® Viral RNA Mini QiagenCatalog #52906
Pipet 560μL prepared Buffer AVL containing linear polyacrylamide into a 1.5 ml microcentrifuge tube.
* Note: If the sample volume is larger than 140 μl, increase the amount of Buffer AVL–linear polyacrylamide proportionally (e.g., a 280 μl sample will require 1120 μl Buffer AVL–linear polyacrylamide) and use a larger tube.
Add 140 μl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to the Buffer AVL–LPA in the microcentrifuge tubes. Mix by pulse-vortexing for 15 s. 00:00:15
* Note: To ensure efficient lysis, it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution. Frozen samples that have only been thawed once can also be used.
Incubate at room temperature (15–25°C) for 10 min.
* Note: Viral particle lysis is complete after lysis for 10 min at room temperature. Longer incubation times have no effect on the yield or quality of the purified RNA.
00:10:00
Briefly centrifuge the tube to remove drops from the inside of the lid.
Add 560 μl ethanol (96–100%) to each sample, and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid. 00:00:15
* Note: Use only ethanol, since other alcohols may result in reduced RNA yield and purity. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. If the sample volume is greater than 140 μl, increase the amount of ethanol proportionally (e.g., a 280 μl sample will require 1120 μl ethanol). To ensure efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution.
Carefully apply 630 μl of the solution from step 8 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate.
* Note: Close each spin column to avoid cross-contamination during centrifugation.
* Note: Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise. Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the solution has not completely passed through the membrane, centrifuge again at a higher speed until all of the solution has passed through.
8000 rpm, 00:01:00
Carefully open the QIAamp Mini column, and repeat step 9. If the sample volume was greater than 140 μl, repeat this step until all of the lysate has been loaded onto the spin column.
Carefully open the QIAamp Mini column, and add 500 μl Buffer AW2. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with step 14, or to eliminate possible Buffer AW2 carryover, perform step 13 and then continue.
* Note: Residual Buffer AW2 in the eluate may cause problems in downstream applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow- through, containing Buffer AW2, contacting the QIAamp Mini column.
14000 rpm, 00:03:00
Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not provided), and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.14000 rpm, 00:01:00
Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not provided). Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 30 μl DNase/RNase Free Water equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min.800
Centrifuge at 6000 x g (8000 rpm) for 1 min.
8000 rpm, 00:01:00
Transfer 30µL of eluted nucleic acid to a new microcentrifuge tube.
DNase Treatment
DNase Treatment
Create a master mix of TURBO DNase Mix:
Add 1 μL TURBO DNase (2 U) (for up to 10 μg RNA in a 50 μL reaction), 5µL TURBO DNase Buffer, and 14µL of H2O per sample.
TURBO™ DNase (2 U/µL)Thermo FisherCatalog #AM2238
Add 20µL of TURBO DNase Mix to 30µL of sample.
Incubate at 37°C for 30 minutes. 37 °C
00:30:00
Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
LINK
Any heat block will suffice
SPECIFICATIONS
Zymo Clean-up and Concentrator
Zymo Clean-up and Concentrator
Add 2 volumes RNA Binding buffer to each sample (ex. 100µL buffer + 50 µL sample)
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Add an equal amount of 100% ethanol and mix. (ex. 150µL ethanol)
Transfer the sample to the Zymo-Spin IC Column in a Collection Tube and centrifuge for 30 seconds. Discard the flow-through
* For samples >800 µL, Zymo-Spin columns can be reloaded
14000 rpm, 00:00:30
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
Add 400 µL RNA Prep Buffer to the column and centrifuge at 10,000g– 16,000g for 30 seconds. Discard flow-through.
14000 rpm, 00:00:30
Add 700 µL RNA Wash Buffer to the column and centrifuge at 10,000g– 16,000g for 30 seconds. Discard flow-through.
14000 rpm, 00:00:30
Centrifuge at 10,000g– 16,000g for 2 minutes to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase free tube (Not provided in RNA Clean & Concentrator-5 kit).
14000 rpm, 00:02:00
Add 8-10 µL Nuclease-Free water directly to the column matrix and centrifuge at 10,000g– 16,000g for 30 seconds. The eluted RNA can be used immediately or stored at -70°C.
SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition
SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition
Make a working stock of your primer A. Stock: 100pmol/1µL. Add 4µL of stock + 6µL H2O. You now have a 10µL of a 40pmol/µL working stock.
Add 1 µL Primer A working stock to 4 µL extracted RNA. Heat to 65°C for 5 minutes in a thermocycler and let cool at 4°C for 5 minutes.
Equipment
SimpliAmp Thermal Cycler
NAME
PCR
TYPE
Applied Biosystems
BRAND
A24811
SKU
LINK
Any standard PCR thermocycler will suffice
SPECIFICATIONS
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 2 μL 5X RT buffer, 1 μL 10 mM dNTP, 1 μL water, 0.5 μL 0.1M DTT, and 0.5 μL SSIV RT.
Add 5 µL master mix to reaction. Incubate at 50°C for 10 minutes.
50 °C 00:10:00
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 1 μL 5X Sequenase buffer, 3.8 μL water, and 0.15 μL Sequenase.
After 10 minute incubation, Add 5 µL of Sequenase Mix #1 to the reaction.
Incubate at 37°C for 8 min.
37 °C 00:08:00
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 0.45 μL Sequenase dilution buffer, and 0.15 μL Sequenase.
After 8 min incubation, add 0.6µL of Sequenase Mix #2 to the reaction.
Incubate at 37°C for 8 min.
37 °C 00:08:00
After 8 min incubation, add 1µL of RNase H to each sample.
Incubate reaction at 37°C for 20 min.
37 °C 00:20:00
* Round A is now complete and samples can be stored at -20°C
SISPA B: PCR Amplification of Randomly Primed cDNA cDNA Amplification
SISPA B: PCR Amplification of Randomly Primed cDNA cDNA Amplification
Make a master mix for 1 reaction (scale up as needed) consisting of 5 µL AccuTaq LA 10x Buffer, 2.5 µL dNTP mix, 1µL DMSO, 0.5 µL AccuTaq LA DNA Polymerase, 35 µL nuclease free water, and 1 µL Primer B.
*Do not dilute Primer B
Add 5 µL of product from SISPA Round A to 45 µL master mix.
Run PCR with the following conditions 98°C for 30s, followed by 30 cycles of 94°C for 15 s, 50°C for 20 s, and 68°C for 2 min, a final step of 68°C for 10 min, and then a 4°C hold until you’re ready for the cleanup.
* Can freeze in the -20°C until ready
Equipment
SimpliAmp Thermal Cycler
NAME
PCR
TYPE
Applied Biosystems
BRAND
A24811
SKU
LINK
Any standard PCR thermocycler will suffice
SPECIFICATIONS
Bead Clean-up
Bead Clean-up
Amplified cDNA was purified using a 1:1 ratio of AMPure XP beads. Add 50µL of resupsended AMPure XP beads to 50µL SISPA product.
Incubate at RT for 10 minutes
Room temperature 00:10:00
Spin down briefly
Equipment
Mini-centrifuge
NAME
Centrifuge
TYPE
Fisher
BRAND
S67601B
SKU
LINK
Any standard mini centrifuge with adapters for different tube sizes will suffice
SPECIFICATIONS
Place on magnet. Remove and discard supernatant once solution turns clear. Be sure not to disturb beads.
Equipment
Magnetic Stand
NAME
Magnetic Stand
TYPE
Thermo Scientific
BRAND
MR02
SKU
LINK
Any magnetic rack that fits your tubes will suffice.
SPECIFICATIONS
Wash with 200µL of 70% EtOH.
Remove EtOH without disturbing beads.
Repeat wash. Remove EtOH without disturbing beads.
Remove samples from magnetic rack and spin down briefly.
Remove residual ethanol
Let air dry briefly - Do not overdry bead pellet
Resuspend sample in 50µL of H2O.
Incubate samples at RT for 5 minutes.
Room temperature 00:05:00
Transfer tubes to magnetic rack. Transfer 48µL of eluted product to a new tube.
Continue onto library prep protocol of your choice.