Viral metagenomics using SMART-9n amplification and nanopore sequencing
Oct 03, 2019
Open access
Protocol CitationIngra Claro Morales, Josh Quick 2019. Viral metagenomics using SMART-9n amplification and nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.7w5hpg6
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: Oct 03, 2019
Last Modified: Oct 03, 2019
PROTOCOL integer ID: 28349

Public workspaceViral metagenomics using SMART-9n amplification and nanopore sequencing

  • 1Universidade de São Paulo;
  • 2University of Birmingham
Abstract
This is a SMART-Seq (Switch Mechanism at the 5′ End of RNA Templates) protocol developed to use random 9n priming and be compatible with ONT RLB rapid, barcoded adapters available from the SQK-RPB004 kit. We have generated cDNA reads in excess of 10 kb from Zika viral reference material with this protocol.

MATERIALS
Reagents required:

Ultrafree-MC Centrifugal Filter (Merck, 10228490)
ZymoBIOMICS DNA Microprep Kit with Lysis Tubes (Zymo, D4301)
Quick-RNA Viral Kit (Zymo, R1034)
Turbo DNase (Thermo Fisher, AM2238)
Clean up kit Zymo Research, (Zymo, R1015)
SuperScript IV (Thermo Fisher, 15307696)
RNase OUT (Thermo Fisher, 10777019)
Ampure XP 60 ml (Beckman Coulter, A63881)
LongAmp Taq 2X Master Mix (NEB, M0287)

Oligos required:

RLB TSO (Order as RNA oligo from IDT):
GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATrGrGrG
RLB RT 9N:
TTTTTCGTGCGCCGCTTCAACNNNNNNNNN
RLB PCR (not required but useful for testing):
TTTTTCGTGCGCCGCTTCA

Centrifugal filtration
1
Transfer up to Amount500 µL sample directly onto a Ultrafree-MC Centrifugal Filter column

2
Spin at Centrifigation5000 x g for Duration00:01:00

3
Recover filtrate into Amount1.5 mL Eppendorf tube

4
Remove basket and discard
5
Close lid and place on ice
Viral RNA extraction
6
In a Amount2 mL Eppendorf tube combine Amount200 µL sample,Amount200 µL DNA/RNA Shield (2x concentrate) and mix well by pipetting

7
Add Amount800 µL Viral RNA Buffer and mix well by pipetting

8
Load Amount600 µL onto a column in a collection tube and spin at Centrifigation10000 x g for Duration00:00:15 discard flow through, place in a new collection tube

9
Add Amount500 µL Viral wash buffer and spin at Centrifigation10000 x g for Duration00:00:15 , discard flow through

10
Add Amount500 µL 100% ethanol and spin at Centrifigation10000 x g for Duration00:01:00 , discard flow through, place in a clean Amount1.5 mL tube

11
Add Amount15 µL of DNA/RNA-Free Water and incubate at RT for Duration00:03:00

12
Spin at Centrifigation10000 x g for Duration00:00:15

DNase Treatment
13
Set heat block to Temperature37 °C

14
Set up the following reaction:

Component Volume

RNA Amount44 µL
10X TURBO DNase Buffer Amount5 µL
TURBO DNase Amount1 µL
Total Amount50 µL

15
Incubate at Temperature37 °C for Duration00:30:00

DNase cleanup
16
Add Amount100 µL RNA Binding Buffer and mix by vortexing Duration00:00:05 and spin down

17
Add Amount150 µL 100% ethanol and mix by vortexing for Duration00:00:15 and spin down

18
Transfer Amount300 µL to a Zymo-Spin IC column in a Amount2 mL collection tube and spin at Centrifigation6000 x g for Duration00:00:15 discard flow through

19
Add Amount400 µL RNA Prep Buffer and spin at Centrifigation6000 x g for Duration00:00:15 , discard flow through

20
Add Amount700 µL RNA Wash Buffer and spin at Centrifigation6000 x g for Duration00:00:15 , discard flow through and place in a new Amount1.5 mL Eppendorf tube

21
Add Amount10 µL DNase/RNase Free Water and incubate at RT for Duration00:01:00

22
Spin at Centrifigation6000 x g for Duration00:00:15

23
Label as ‘Viral RNA’ and place on ice
Viral DNA extraction
24
Set heat block to Temperature55 °C

25
In a Amount2 mL Eppendorf tube combine Amount200 µL sample, Amount200 µL DNA/RNA Shield (2X concentrate), Amount20 µL Proteinase K and mix well by pipetting

26
Incubate at Temperature55 °C for Duration00:30:00


27

Set heat block to Temperature60 °C

28
Add Amount1.200 mL binding buffer and mix well

29
Load Amount800 µL onto a Zymo-Spin IIC-Z column in a collection tube and spin at Centrifigation8000 x g for Duration00:00:15 , discard the discard flow-through and reload as many times as required

30
Transfer to to a new collection tube add Amount400 µL DNA Wash Buffer 1 and spin at Centrifigation8000 x g for Duration00:00:15 , discard flow-through

31
Add Amount700 µL DNA Wash Buffer 2 and spin at Centrifigation8000 x g for Duration00:00:15 .

32
Add Amount200 µL DNA Wash Buffer 2 and spin at Centrifigation8000 x g for Duration00:01:00 .


33
Transfer to a new Amount1.5 mL Eppendorf tube and Amount50 µL DNA/RNA Free Water preheated to Temperature60 °C to the column, incubate at RT for Duration00:01:00 .

34
Spin at Centrifigation8000 x g for Duration00:01:00 .

SMART-9n amplification
35
Combine the following in a 0.2ml 8-strip tube.

Component Volume

RLB RT 9N (2 uM) Amount1 µL
dNTPs (10 mM ea.) Amount1 µL
Template RNA Amount10 µL
Total Amount12 µL

36
Mix and spin down.
37
Incubate at Temperature65 °C for Duration00:05:00 then snap cool on ice.

38
Make up the following master mix and add Amount8 µL to the Amount12 µL annealed RNA:

Component Volume

SSIV buffer (5x) Amount4 µL
DTT (100 mM) Amount1 µL
RNase OUT Amount1 µL
SS IV RTase (200 U/ul) Amount1 µL
RLB TSO (2 uM) Amount1 µL
Total Amount20 µL

39
Start the following program on a thermocycler:

Temperature42 °C for Duration01:30:00
Temperature70 °C for Duration00:10:00

40
Set up the following reaction to amplify cDNA in a new 0.2ml 8-strip tube:

Component Volume

LongAmp Taq 2X master mix Amount25 µL
RLB (01-12) Amount0.5 µL
NFW Amount19.5 µL
cDNA Amount5 µL
Total Amount50 µL

41
Start the following program on the thermal cycler:

Step Temperature Time Cycles

Heat Activation Temperature95 °C Duration00:00:45 1
Denaturation Temperature95 °C Duration00:00:15 26
Annealing Temperature56 °C Duration00:00:15 26
Extension Temperature65 °C Duration00:05:00 26
Final extension Temperature65 °C Duration00:10:00 1
Hold Temperature4 °C Indefinite 1

PCR clean-up
42
Clean up products with 1x Ampure XP and elute in 30 ul EB
Protocol
Amplicon clean-up using SPRI beads
NAME

Amplicon clean-up using SPRI beads

CREATED BY
Josh Quick

42.1
Critical
Vortex SPRI beads thoroughly to ensure they are well resuspended, the solution should be a homogenous brown colour.

ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880

42.2
Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add Amount50 µL SPRI beads to a Amount50 µL reaction.

42.3
Pulse centrifuge to collect all liquid at the bottom of the tube.

42.4
Incubate for Duration00:05:00 at room temperature.

42.5
Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear.

42.6
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
42.7
Add Amount200 µL of room-temperature Concentration70 % volume ethanol to the pellet.



42.8
Carefully remove and discard ethanol, being careful not to touch the bead pellet.
42.9
Go togo to step #42.7 and repeat ethanol wash.

42.10
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
42.11
With the tube lid open incubate for Duration00:01:00 or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

42.12
Resuspend pellet in Amount30 µL Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for Duration00:02:00 .
ReagentElution Buffer (EB)QiagenCatalog #19086



42.13
Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
42.14
Quantify Amount1 µL product using the Quantus Fluorometer using the ONE dsDNA assay.
ReagentQuantiFluor(R) ONE dsDNA System, 100rxnPromegaCatalog #E4871



QC
43
Quantify the PCR products using the Quantus Fluorometer using the ONE dsDNA assay.
Protocol
DNA quantification using the Quantus fluorometer
NAME

DNA quantification using the Quantus fluorometer

CREATED BY
Josh Quick


43.1
Remove Lambda DNA 400 ng/µL standard from the freezer and leave on ice to thaw. Remove ONE dsDNA dye solution from the fridge and allow to come to room temperature.

ReagentQuantiFluor(R) ONE dsDNA System, 500rxnPromegaCatalog #E4870



43.2
Set up two Amount0.5 mL tubes for the calibration and label them 'Blank' and 'Standard'

43.3
Add Amount200 µL ONE dsDNA Dye solution to each tube.
43.4
Mix the Lambda DNA standard 400 ng/µL standard by pipetting then add Amount1 µL to one of the standard tube.

43.5
Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.
43.6
Allow both tubes to incubate at room temperature for Duration00:02:00 before proceeding.

43.7
Selection 'Calibrate' then 'ONE DNA' then place the blank sample in the reader then select 'Read Blank'. Now place the standard in the reader and select 'Read Std'.
43.8
Set up the required number of Amount0.5 mL tubes for the number of DNA samples to be quantified.
Note
Use only thin-wall, clear, 0.5mL PCR tubes such as Axygen #PCR-05-C


43.9
Label the tubes on the lids, avoid marking the sides of the tube as this could interfere with the sample reading.
43.10
Add Amount199 µL ONE dsDNA dye solution to each tube.

43.11
Add Amount1 µL of each user sample to the appropriate tube.
Note
Use a P2 pipette for highest accuracy.


43.12
Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.

43.13
Allow all tubes to incubate at room temperature forDuration00:02:00 before proceeding.

43.14
On the Home screen of the Quantus Fluorometer, select `Protocol`, then select `ONE DNA` as the assay type.
Note
If you have already performed a calibration for the selected assay you can continue, there is no need to perform repeat calibrations when using ONE DNA pre diluted dye solution. If you want to use the previous calibration, skip to step 11. Otherwise, continue with step 9.

43.15
On the home screen navigate to 'Sample Volume' and set it to Amount1 µL then 'Units' and set it to ng/µL.

43.16
Load the first sample into the reader and close the lid. The sample concentration is automatically read when you close the lid.
43.17
Repeat step 16 until all samples have been read.
43.18
The value displayed on the screen is the dsDNA concentration in ng/µL, carefully record all results in a spreadsheet or laboratory notebook.
Rapid adapter attachment
44
Pool all barcoded products to a total of Concentration200 Femtomolar (fM) in Amount10 µL of Concentration10 millimolar (mM) Tris-HCl pH 8.0 with Concentration50 millimolar (mM) NaCl
45
Add Amount1 µL RAP adapter and mix by pipetting, incubate at RT Duration00:05:00

MinION sequencing
46
Prime the flowcell and load sequencing library onto the flowcell.
Protocol
Priming and loading a MinION flowcell
NAME

Priming and loading a MinION flowcell

CREATED BY
Josh Quick

46.1
Thaw the following reagents at room temperature before placing on ice:

Sequencing buffer (SQB)
Loading beads (LB)
Flush buffer (FLB)
Flush tether (FLT)
46.2
Add Amount30 µL FLT to the FLB tube and mix well by vortexing.

46.3
If required place a new MinION flowcell onto the MinION by flipping open the lip and pushing one end of the flowcell under the clip and pushing down gently.

46.4
Rotate the inlet port cover clockwise by 90° so that the priming port is visible.
46.5
Take a P1000 pipette and tip and set the volume to Amount800 µL . Place the tip in the inlet port and holding perpendicularly to the plane of the flowell remove any air from the inlet port by turning the volume dial anti-clockwise.
Note
Be careful not to remove so much volume that air is introduced onto the rectangular array via the outlet.


46.6
Load Amount800 µL of FLB (plus FLT) into the flow cell via the inlet port, dispense slowly and smoothly trying to avoid the introduction of any air bubbles.

46.7
Wait forDuration00:05:00 .

46.8
Gently lift the SpotON cover to open the SpotON port.
46.9
Load another Amount200 µL of FLB (plus FLT) into the flow cell via the inlet port, this will initiate a siphon at the SpotON port to allow you to load the library dilution.
46.10
In a new tube prepare the library dilution for sequencing:

Component Volume

SQB Amount37.5 µL
LB Amount25.5 µL
Final library Amount12 µL
Total Amount75 µL

Note
Mix LB immediately before use as they settle quickly.

Dilute library in EB if required.

46.11
Mix the prepared library gently by pipetting up and down just prior to loading.
46.12
Add the Amount75 µL library dilution to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop siphons into the port before adding the next.

46.13
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the inlet port and close the MinION lid.