TITLE: Lloviu cuevavirus sequencing protocol
AUTHORS: Gábor Tóth, Gabor Kemenesi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ARTIC-like amplicon sequencing protocol for MinION for Lloviu cuevavirus</div><div class = "text-block">The increasing risk of emerging infectious diseases (EIDs) for humanity is a constantly growing problem. Next-generation sequencing rapidly increased the discovery of distant filovirus species worldwide. Lloviu virus is currently the only known member of Filoviridae family found in Europe. We prepared and tested this protocol on Hungarian Lloviu positive native samples found in our laboratory. We are aware of the extreme rarity of LLOV, we provide this protocol to facilitate research attempts and widen the possibilities of future studies.</div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components in an 0.2mL 8-strip tube;Component                                        Volume50µM random hexamers                 10mM dNTPs mix (10mM each)    Template RNA                                   Total
1 µl
1 µl
11 µl
13 µl
Viral RNA input from a native sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
A mastermix should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows: for Place on ice for
65 °C
?. [cDNA preparation]
Add the following to the annealed template RNA: Component	                                Volume SSIV Buffer	                                 100mM DTT                                  RNaseOUT RNase Inhibitor 	 SSIV Reverse Transcriptase	 Total
4 µl
1 µl
1 µl
1 µl
20 µl
A mastermix should be made up in the mastermix cabinet and added to the denatured RNA in the extraction and sample addition cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows:Hold at
25 °C
42 °C
70 °C
5 °C
?. [Primer pool preparation]
If required resuspend lyophilised primers at a concentration of 100µM each
Three different primer set were designed for this protocol with the usage of Primal Scheme. These original sets were updated wit alternative primers based on the generated sequence data and empirical experiences.1, LLOV_400bpThis set generates 400 basepair long amplicons with ~75 basepair overlaps.2, LLOV_500bpThis set generates 500 basepair long amplicons with ~50 basepair overlaps.3, LLOV_2000bpThis set generates 2000 basepair long amplicons with ~130 basepair overlaps. These primers were choosen from LLOV_400bp set and reutilised here as a new .
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each odd region primer to a  Eppendorf labelled “Pool 1 (100µM)” and each even region primer to a  Eppendorf labelled  “Pool 2 (100µM)”. These are your 100µM stocks of each primer pool.Final volume of the 100µM stocks:LLOV_400bp Pool 1:   405 µl     (81 primer) LLOV_400bp Pool 2:   410 µl     (82 primer)LLOV_500bp Pool 1:  290 µl      (58 primer)LLOV_500bp Pool 2:  260 µl      (52 primer)LLOV_2000bp Pool 1:   50 µl     (10 primer)LLOV_2000bp Pool 2:   50 µl     (10 primer)
5 µl
1.5 mL
1.5 mL
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Primers need to be used at a final concentration of 0.015µM per primer.
?. [Multiplex PCR]
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:LLOV_400bpComponent                                             Pool 1                Pool 2                 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             V3 Primer Pool 1 or 2 (10µM)               Nuclease-free water                              Total                                                         LLOV_500bpComponent                                             Pool 1                Pool 2                 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             V3 Primer Pool 1 or 2 (10µM)               Nuclease-free water                              Total                                                         LLOV_2000bpComponent                                             Pool 1                Pool 2                 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             V3 Primer Pool 1 or 2 (10µM)               Nuclease-free water                              Total
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
3 µl
3 µl
13.75 µl
13.75 µl
22.5 µl
22.5 µl
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
2 µl
2 µl
14.75 µl
14.75 µl
22.5 µl
22.5 µl
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
2 µl
2 µl
14.75 µl
14.75 µl
22.5 µl
22.5 µl
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [Multiplex PCR]
In the extraction and sample addition cabinet add  cDNA to each tube and mix well by pipetting.
2.5 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35 Annealing                   25-35Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35.
?. [PCR clean-up]
Label a  Eppendorf tube for each sample and assemble the following PCR dilution for each sample:Component                                        VolumePool 1 PCR reaction                        Pool 2 PCR reaction                       Nuclease-free water
1.5 mL
2.5 µl
2.5 µl
45 µl
The PCR post-clean up concentration is typically around  . This assumption means we can make a significant saving in terms of protocol time for only a slight loss in barcode representation. To get a better result you should use multiple primer sets (LLOV_400bp, LLOV_500bp, LLOV_2000bp) with different barcodes. This can be useful in later bioinformatic analysis (i.e. read length filtering).
?. [Quantification and normalisation]
Label another  Eppendorf tube for each sample.
1.5 mL
Input to the one-pot native barcoding reaction is 50ng per sample. Process at least 6 samples plus one negative control per library in order to have sufficient material to load on the sequencer at the end.
?. [Native barocoding]
Barcode the amplicon pools using the one-pot native barcoding approach.
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?. [Native barocoding]
Quantify  of the barcoded amplicon pool using the Quantus Fluorometer using the ONE dsDNA assay.
1 µl
?. [Native barocoding]
Set up the following AMII adapter ligation and clean-up with SFB.
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?. [Native barocoding]
Quantify  of the final library using the Quantus Fluorometer using the ONE dsDNA assay.
1 µl
Final library can be now be stored in 10 mM Tris pH 8 at 4°C for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
20 ng
From experience we know 20 ng is optimum loading input for short amplicons.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.Our result with different primer sets: