TITLE: SARS-CoV-2 McGill Nextera Flex sequencing protocol_SS_V3_LA1_5uLRT
AUTHORS: Sarah Reiling, Marie-Michelle Simon, Anne-Marie Roy, Shu-Huang Chen, Josh Quick, Ioannis Ragoussis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">How the Nextera DNA Flex Assay Works </span></div><div class = "text-block">The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. A subsequent Sample Purification Beads (SPB) cleanup step then purifies libraries for use on an Illumina sequencer. The double-stranded DNA library is denatured before hybridization of the biotin probe oligonucleotide pool. </div><div class = "text-block"><span style = "font-weight:bold;">PCR Amplicons for Nextera Flex</span></div><div class = "text-block"><span>When starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries < 500 bp. </span><span style = "font-weight:bold;">Therefore, Illumina recommends that amplicons < 500 bp undergo a 1.8 x sample purification bead volume ratio to supernatant during Clean Up Libraries on page 11. </span><span>Shorter amplicons can otherwise be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end. </span></div><div class = "text-block"><span>More information can be found here: </span><a href="https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf</span></a></div></div>

[STEPS]
?. [cDNA preparation]
cDNA PREPARATIONMix the following components in an 0.2mL 8-strip tube;Component                                        Volume50 µM random hexamers                 10 mM dNTPs mix (10 mM each)    Template RNA                                   Total
1 µl
1 µl
11 µl
13 µl
Viral RNA input from a clinical 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
42 °C
70 °C
5 °C
?. [Primer pool preparation]
PRIMER POOL PREPARATIONIf required resuspend lyophilised primers at a concentration of 100 µM each
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each primer pair to a  Eppendorf labelled either “Pool 1 (100 µM)” or “Pool 2 (100 µM)”. Total volume should be  for Pool 1 (100 µM) and  for Pool 2 (100 µM). These are your 100µM stocks of each primer pool.Make another primer pool named "Pool LA1 (100 µM)" that contains 5 µl of primer pairs 5, 17, 23, 26, 66, 70, 74, 91, 97, and 10 ul of primer pair 64.
5 µl
1.5 mL
490 µl
490 µl
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 10 µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination. LA1 primer pool will be diluted to 1 µM primer stock.
Primers need to be used at a final concentration of 0.015 µM per primer. In this case both pools have 98 primers in so the requirement is 3.65 µL primer pools (10 uM) per 25 µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
MULTIPLEX PCRIn the extraction and sample addition cabinet add  RT product to each tube and mix well by pipetting.
5 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:Component                                                             Pool 1 [10 uM primer]        Pool 2 [10 uM]       Pool LA1 [1 uM]Q5 Hot Start High-Fidelity 2X Master Mix         Primer Pool 1 or 2 (10µM pool 1+2; 1µM LA1) Nuclease-free water                                             Total                                                                        Add 5 ul RT product as mentioned in step 10.
12.5 µl
12.5 µl
12.5 µl
3.7 µl
3.7 µl
3.7 µl
3.8 µl
3.8 µl
3.8 µl
20 µl
20 µl
20 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]
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              36 Annealing                   36Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 35
?. [PCR clean-up]
PCR CLEANUPCombine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single Eppendorf tube. Keep Pool LA1 separate from the combined Pool 1+2 until after the clean-up!!
1.5 mL
?. [PCR clean-up]
Clean-up the amplicons using the following protocol:Add an equal volume (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.Incubate for 5 min at room temperature. Pellet on magnet for 5 min. Remove supernatant.Add 200 ul of 80% ethanol to the pellet and wash twice. Elute in 30 ul elution buffer.
Amplicon clean-up should be performed in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Amplicon Quantification and normalisation]
AMPLICON QUANTIFICATION AND NORMALIZATIONQuantify the amplicon pools using a fluorimetric dsDNA assay.  We expect following concentrations:Pool 1+2 combined:100-150 ng/ul for Ct 14-2430-80 ng/ul for Ct 25-2910-30 ng/ul for Ct 30-36Pool LA1:1-10 ng/ul for all Ct
?. [Amplicon Quantification and normalisation]
After quantification of Pool 1+2 and Pool LA1, mix them together in following ratio: 89.8% Pool 1+2 and 10.2% Pool LA1. For this, take a new plate and add 135 ng of Pool 1+2 and 15.3 ng of Pool LA1, and add up with nuclease-free water to a total volume of 30 ul (= 150 ng or 5 ng/ul).
?. [Amplicon Quantification and normalisation]
Use the 30 ul with 150 ng DNA for Nextera Flex Tagmentation. Protocol can be found here:https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdfStart on page 7 with the Tagmentation.
?. [Tagment Genomic DNA]
For automated protocol : at step 7, 22 ul tagmentation master mix is transfered.
?. [Post Tagmentation Cleanup]
?. [Amplify Tagmented DNA]
For automated protocol : at step 9,  44 ul tagmentation master mix is transfered.
?. At step 14, beads can be resuspended in 62 uL RSB , followed by a 60 uL transfer of supernatant at step 18.  This is used to decrease the concentration of the final pool in order to facilitate the QC step.
?. [Pool Libraries]
?. [Check Library Quality]
?. [Dilute Libraries to the Starting Concentration]
?. [Primer pool preparation]
ARTIC nCov-2019  only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below. https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed