TITLE: SARS-CoV-2 Whole Genome Sequencing on Illumina - Tiling PCR
AUTHORS: Guerrino Macori, Seamus Fanning

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently obtaining, through the amplicons tiling, the whole viral genome using V3 nCov-2019 primers (ARTIC). This is followed by library construction and pooling of samples and quantitation, prior to sequencing on the Illumina MiSeq. </div><div class = "text-block"><span>The SOP is adapted from the nCoV-2019 sequencing protocol: </span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w</span></a><span>, and it was used in this study:</span></div><div class = "text-block">Lucey M, Macori G, Mullane N, Sutton-Fitzpatrick U, Gonzalez G, Coughlan S, Purcell A, Fenelon L, Fanning S, Schaffer K. Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks. Clinical Infectious Diseases. 2020.</div><div class = "text-block"><a href="https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421</span></a></div></div>

[STEPS]
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
cDNA/Reverse Transcription Section        Date/Initials:_________________In this section, the nucleic acid extracted and used for the qPCR diagnostic test is used as starting material for the sequencing.
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  In a PCR hood, mix the following reagents in a  PCR tube set or PCR plate:  ABC1ReagentVolume (µL)MM for N+2 samples260 µM random hexamers1.0310 mM dNTPs mix (10 mM each)1.04Template RNA 11.05Total13.0Lot# _______________ Exp. Date _______________Lot# _______________ Exp. Date _______________
0.2 mL
ABC1ReagentVolume (µL)MM for N+2 samples260 µM random hexamers1.0310 mM dNTPs mix (10 mM each)1.04Template RNA 11.05Total13.0
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. Each reaction should have   when mixed. If using master mix, it is recommended to add the   of the master mix to the PCR tube first, then add the 11 µl of RNA to help prevent contamination.
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Mix gently and briefly centrifuge to spin down the components, and return  .
on ice
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Preheat Thermocycler to , with heated lid at
65 °C
105 °C
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Incubate the reaction at  for  00:05:00, followed by an immediate snap-cool  for at least .
65 °C
on ice
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  In a clean  LoBind tube (96 well plates can also be used), mix together the following reagents:  ABC1ReagentVolume  (uL)MM for N+2 samples2SuperScript IV RT 5X Buffer4.03 100mM DTT  1.04RNaseOUT RNase Inhibitor1.05Superscript IV Reverse Transcriptase1.06Total7.0Lot# _______________ Exp. Date _______________Lot# _______________ Exp. Date _______________
1.5 mL
ABC1ReagentVolume  (uL)MM for N+2 samples2SuperScript IV RT 5X Buffer4.03 100mM DTT  1.04RNaseOUT RNase Inhibitor1.05Superscript IV Reverse Transcriptase1.06Total7.0
The 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.
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Add the above mastermix () to the annealed DNA () giving a total volume
7 µl
13 µl
20 µl
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Cap the tube (or seal the plate), mix and then briefly centrifuge the contents.
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Preheat thermocycler to  , with heated lid at
42 °C
105 °C
?. [SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription]
[   ]  Incubate sample using the following reverse transcription program:  ABCD1StepTemperature (°C)TimeCycle2Reverse Transcription4250:0013RT Inactivation7010:0014Cool4HoldHoldPAUSE POINT cDNA can be stored at  (same day) or  (up to a week).
ABCD1StepTemperature (°C)TimeCycle2Reverse Transcription4250:0013RT Inactivation7010:0014Cool4HoldHold
4 °C
-20 °C
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR ]
Tiled PCR Section        Date/Initials:_________________This section outlines the process for the tiled PCR approach from the ARTIC protocol. If required resuspend lyophilised primers at a concentration of 100 µM each.Prepare the primer working solution diluting to  using  TE buffer.
Primer pool sequences (v3) can be found here: https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR ]
[   ]  Set up two individual reactions using primer pool 1 (set 1) and primer pool 2 (set 2) in  PCR tubes according to the following table:  ABCDE1ReagentPool 1 (uL)MM for N+2 samplesPool 2 (uL)MM for N+2 samples2Q5 Hot Start HiFi 2x MM12.512.53Primer pool at 10uM (1 or 2)3.73.74Nuclease-free water6.36.35Total22.522.5Lot# _______________ Exp. Date _______________
0.2 mL
ABCDE1ReagentPool 1 (uL)MM for N+2 samplesPool 2 (uL)MM for N+2 samples2Q5 Hot Start HiFi 2x MM12.512.53Primer pool at 10uM (1 or 2)3.73.74Nuclease-free water6.36.35Total22.522.5
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR ]
[   ]  Aliquotate  from the mastermix into 2 96-well PCR plates or 2 sets of PCR tubes.
22.5 µl
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR ]
[   ]  Add  of sample cDNA (from step 1.9) to each pool giving a total volume  and mix by pipetting.
2.5 µl
25 µl
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR ]
[   ]  Heat seal and place the plates onto a thermocycler and run the following program. Important! Heat seal to minimise evaporation.Note: Amplification should ideally be performed in a different lab to minimise the risk of contaminating other samples.  ABCD1StepTemperatureTimeCycles2Initial Denaturation98°C0:3013Denaturation98°C0:15354Anneal and Extension65°C5:00355Cool4°CHoldHold
ABCD1StepTemperatureTimeCycles2Initial Denaturation98°C0:3013Denaturation98°C0:15354Anneal and Extension65°C5:00355Cool4°CHoldHold
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35
Pause point, Amplified cDNA can be stored at   (overnight) or   up to a week.
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
Section for Clean-Up and Size Selection        Date/Initials:_________________Reagent preparation: Prepare the  ethanol (EtOH) using the following calculation:  x (# Sample + 1: ________________) = ________ mL total volume (EtOH 100%)mL total volume x 0.8 = ________ mL EtOHTotal volume _______mL  - _______mL EtOH = ________mL H2O
0.36 mL
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ]  Combine the entire volumes of primer 1 and primer 2 PCR reactions ( in total) into one clean PCR plate (or  PCR tubes set). Add 0.8X volume of SPRI beads per sample ( SPRI :  amplified cDNA), mix well by pipetting.
50 µl
40 µl
50 µl
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ]  Incubate  at .
0 Room temperature
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Transfer the plate on the magnet and incubate for   at .
0 Room temperature
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Keep the plate on the magnet and remove the superanatant by pipetting from the bottom.
Keep the supernatant in case you have to go back for quality assessment.
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Wash the beads in the magnet with  of freshly prepared 80 % volume EtOH without disturbing the pellet and incubate for  and remove the EtOH.
180 µl
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ]  Repeat previous step (total 2 washes).
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol and allow to dry for approximately .
Do not over-dry the beads. This may result in a lower recovery of DNA
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Remove the plate from the magnet and add  of nuclease-free water, resuspend the beads pipetting up and down at least 10 times or vortex at  for
30 µl
Centrifuge: 1800 33
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Incubate at room temperature for
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ] Transfer the plate on the magnet and incubate for  at
0 Room temperature
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ]  Carefully transfer the supernatant into a new plate, taking care not to disturb the bead pellet.
PAUSE POINT Purified amplified cDNA can be stored at -20°C for several weeks prior to library preparation.
?. [SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection]
[   ]  Quantify the  sample on Qubit fluorometer or similar instrument and store completed PCR amplified cDNA prep
-20 °C
Purified amplified cDNA is quantified with the use of the dsDNA HS Assay kit. 30 uL of samples should contain 50 ng to 1 ug of DNA (optimal 100-500 ng of DNA). If the DNA concentration at this step is less than ~3ng/uL, the sample did not amplify well and it could be under-represented in the final sequencing reaction.To streamline the workflow, the samples are not normalised but used as input for library preparation,   the entire volume is used for the library preparation.To normalise, add enough DNA to reach a total of at least 100 ng** and add molecular grade water to bring the total volume to 30 µl.**NOTE: Preferred amount is 100 ng to 500 ng.  Less than that can lead to under-representation of the sample in the final pool.
?. [NEBNext library preparation protocol - Fragmentation/End prep]
This section is an adaptation protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥
100 ng
For inputs 100 ng, size selection is not recommended. For 100 ng inputs, either the no size selection protocol or a size selection protocol can be followed.
Starting Material100–500 ng purified DNA. If the input DNA is less than 26 µl, add molecular grade water  or 1X TE (  Tris  ,    EDTA to a final volume of  .
?. [NEBNext library preparation protocol - Fragmentation/End prep]
[   ]  Prepare enzyme Master Mix using the following table:  ABC1ReagentVolume (uL)*(#samples+2)2NEBNext Ultra II FS Reaction Buffer 7 µl3NEBNext Ultra II FS Enzyme Mix 2 µl4Total Volume9 µl
ABC1ReagentVolume (uL)*(#samples+2)2NEBNext Ultra II FS Reaction Buffer 7 µl3NEBNext Ultra II FS Enzyme Mix 2 µl4Total Volume9 µl
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place on ice until use.Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place on ice.
?. [NEBNext library preparation protocol - Fragmentation/End prep]
[   ]  Add   of purified DNA to the mix. Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.
26 µl
?. [NEBNext library preparation protocol - Fragmentation/End prep]
[   ]  In a Thermocycler, with the heated lid set to 75°C, run the following program:  ABC1StepTempTime2137°C10 min3265°C30 min4Hold4°CHold
ABC1StepTempTime2137°C10 min3265°C30 min4Hold4°CHold
OPTIMIZATIONFragmentation occurs during the 37°C incubation step. Use the chart below to determine the incubation time required to generate the desired fragment sizes. Incubation time may need to be optimized for individual samples. Run the fragmented suspension on Bioanalyzer to visualize the size distribution.  ABC1Fragmentation sizeIncubation at 37°COptimization2&nbsp;100 bp-250 bp&nbsp;30 min&nbsp;30-40 min3&nbsp;150 bp-350 bp&nbsp;20 min&nbsp;20-30 min4&nbsp;200 bp-450 bp&nbsp;15 min&nbsp;15-20 min5&nbsp;300 bp-700 bp&nbsp;10 min&nbsp;5-15 min6&nbsp;500 bp-1 kb&nbsp;5 min&nbsp;5-10 minNEBNext End Prep                                                 Vol/PCR RXN (µl)  NEBNext Ultra II End Prep Enzyme Mix              1.2  NEBNext Ultra II End Prep Reaction Buffer        2.8     Total  	                                                                  4Temperature  	  Time     20°C  	          30 minutes     65°C  	          30 minutes     4°C  	              ∞https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgttjwnn?step=26
If necessary, samples can be stored at –  , however, a slight loss in yield (~20%) may be observed. It is recommend continuing with adaptor ligation before stopping.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Add the following components directly to the FS Reaction Mixture:  AB1ComponentVolume2FS Reaction Mixture (Step 4.3) 35 µl3NEBNext Ultra II Ligation Master Mix 30 µl4NEBNext Ligation Enhancer 1 µl5NEBNext Adaptor for Illumina 2.5 µl6 Total Volume 68.5 µl
AB1ComponentVolume2FS Reaction Mixture (Step 4.3) 35 µl3NEBNext Ultra II Ligation Master Mix 30 µl4NEBNext Ligation Enhancer 1 µl5NEBNext Adaptor for Illumina 2.5 µl6 Total Volume 68.5 µl
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. It is not recommended adding adaptor to a premix in the Adaptor Ligation Step.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Incubate at  for  in a thermocycler with the heated lid off.
20 °C
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Add   µl of USER Enzyme to the ligation mixture from Step 5.1.
3 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Mix well and incubate at  for   with the heated lid set to ≥
37 °C
47 °C
Samples can be stored overnight at
?. [NEBNext library preparation protocol - Adapter ligation]
Size Selection 275-475bp of Adaptor-ligated DNA for DNA Input >100 ng.Volume of SPRIselect for 1st bead selection        Volume of SPRIselect for 2nd bead selection       [   ]  Bring the volume of the reaction up to   by adding    of 0.1% TE Buffer.
25 µl
10 µl
100 µl
28.5 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Add  of the Ampure XP Beads to the  sample and mix well by pipetting up and down.
25 µl
100 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Incubate at room temperature for
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Place the plate on magnetic block for
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Carefully transfer the supernatant ~  into a new wells. (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
125 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Add   of the Ampure XP Beads to the supernatant from step 38. Mix well by pipetting up and down.
10 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Incubate at room temperature for
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Place plate on magnetic block for
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Carefully remove and discard the supernatant that contains unwanted DNA. Be careful not to disturb the beads that contain the desired DNA (Caution: do not discard beads).
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Wash the beads adding  of freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for  , and then carefully remove and discard the supernatant.Be careful not to disturb the beads that contain DNA targets.
200 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Repeat Step 5.14 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]   Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding   TE (dilute 1X TE Buffer 1:10 in water).
17 µl
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least  at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
?. [NEBNext library preparation protocol - Adapter ligation]
[   ]  Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer  to a new PCR tube.
15 µl
Samples can be stored at
?. [NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA]
[   ]  Add the following reagents to each well from step 5.19  AB1ComponentVolume2Adaptor Ligated DNA Fragments (Step 5.19)15 µl3NEBNext Ultra II Q5 Master Mix 25 µl4Index Primer/i7 Primer 5 µl5Universal PCR Primer/i5 Primer 5 µl6Total Volume 50 µl
AB1ComponentVolume2Adaptor Ligated DNA Fragments (Step 5.19)15 µl3NEBNext Ultra II Q5 Master Mix 25 µl4Index Primer/i7 Primer 5 µl5Universal PCR Primer/i5 Primer 5 µl6Total Volume 50 µl
?. [NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA]
[   ]  Set a 100 µl or 200 µl pipette to 40 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
?. [NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA]
[   ]  Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:  ABCD1CYCLE STEPTEMPTIMECYCLES2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds5*4Annealing/Extension65°C75 seconds5Final Extension65°C5 minutes16Hold4°C∞
ABCD1CYCLE STEPTEMPTIMECYCLES2Initial Denaturation98°C30 seconds13Denaturation98°C10 seconds5*4Annealing/Extension65°C75 seconds5Final Extension65°C5 minutes16Hold4°C∞
*Cycle number was determined by size of input DNA ~100ng is 4-5 cycles.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
The volumes of AMPure XP beads are for use with the sample contained in the exact buffer at this step. Allow the beads to warm to room temperature for at least 30 minutes before use. [   ]  Vortex SPRIselect to resuspend.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Add  (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
45 µl
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Incubate samples on bench top for at least   at room temperature.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  After  (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Add  of  freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for  , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
200 µl
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Repeat Step 7.6. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding   of  TE (dilute 1X TE Buffer 1:10 in water).
33 µl
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least   at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
?. [NEBNext library preparation protocol - Clean up of PCR reaction]
[   ]  Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer   to a new PCR tube and store at .
30 µl
-20 °C
?. [Assess Library quality ]
Set up dilutions and standards as laid out in the kit protocol for dsDNA high sensitivity kit.Record Qubit readings before normalization.
In this protocol   of library (  buffer)
?. [Assess Library quality ]
[   ]  Run Samples on a bioanalyser and check that the library shows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size.
tape station D1000 HS   of library in   buffer (ladder     in   buffer for each cartridge)
?. [Assess Library quality ]
[   ]  Calculate the dilutions required to normalise each sample to a 4nM concentration using the following formula:((LibraryConc*(1000)*(1/expected.length)*(1/Average.fragment.length))*1000)
Note: If a peak ~80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer trace, bring up the sample volume (from Step 2.5.11.) to 50 µl with 0.1X TE Buffer and repeat the Cleanup of PCR Reaction in Section 2.5.
?. [Assess Library quality ]
[   ]  Run Samples on a bioanalyser and check that the library sthows a narrow distribution with an expected peak size based on fragmentation time and size selection. Record the the average peak bp size
Calculate the molar concentration of each library to be diluted using average size from the TapeStation and mass from Qubit, using the following equation: (????????????? (??⁄??)) / (660 ?⁄??? × ??? ??????? ???????? ????) × 10^6 ??/? = ????????????? (??)Make a 4nM dilution of each library
?. [MiSeq Sequencing ]
Pooling and Library Denaturation        Date/Initials:_________________This section demonstrates how to generate a pooled library for V3 reagents on the MiSeq.
Thaw the MiSeq reagents overnight or in a RT waterbath.Remove HT1 from freezer and thaw at room temperature. Store at 2°C to 8°C until you are ready to dilute denatured libraries.
Make a fresh dilution of 0.2N of NaOH and Tris by combining the following volumes in a microcentrifuge tube: 800 uL laboratory-grade water 200 stock 1.0N NaOHmicrocentrifuge tube: 800 uL laboratory-grade water 200 stock 1.0M Tris
?. [MiSeq Sequencing ]
[   ]  Pool  of each normalised sample into one eppendorf tube.
5 µl
?. [MiSeq Sequencing ]
[   ]  Combine the following volumes in a microcentrifuge tube:  4nM library and  of 0.2 N NaOH.[   ]  Vortex briefly and then centrifuge at 280 x g for 1 minute. [   ]  Incubate at room temperature for
5 µl
5 µl
?. [MiSeq Sequencing ]
[   ]  Add  of 0.2N TrisHCL and  of pre-chilled HT1 to the tube containing the denatured library. The result is 1 mL of a 20 pM denatured library.
5 µl
985 µl
?. [MiSeq Sequencing ]
[   ]  Dilute the 20 pM library to the desired concentration, see table below:  ABCDEFG1Concentration6 pM8 pM10 pM12 pM15 pM20 pM220 pM library180 uL240 uL300 uL360 uL450 uL600 uL3Pre-chilled HT1420 uL360 uL300 uL240 uL150 uL0 uL[   ]  Invert to mix and then pulse centrifuge
ABCDEFG1Concentration6 pM8 pM10 pM12 pM15 pM20 pM220 pM library180 uL240 uL300 uL360 uL450 uL600 uL3Pre-chilled HT1420 uL360 uL300 uL240 uL150 uL0 uL
?. [MiSeq Sequencing ]
[   ]  Dilute stock PhiX to 4nM by combining: 2 uL 10 nM PhiX library 3 uL 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20
?. [MiSeq Sequencing ]
[   ]  Mix reagents of the MiSeq cartridge thoroughly by inverting several times.[   ]  Using a fresh 1000 uL pipette tip, transfer the denatured and library (with PhiX spiked) into position 17.
?. [MiSeq Sequencing ]
[   ]  Load the sample sheet and reagents according to onscreen instructions in the MiSeq Control software.
?. [MiSeq Sequencing ]
[   ]  Vortex briefly to mix and centrifuge at 280 x g for 1 minute. [   ]  Incubate at room temperature for 5 minutes
?. [MiSeq Sequencing ]
Denature the PhiX control by adding the following volumes in a microcentrifuge tube: 5 uL 4nM PhiX library 5 uL 0.2N NaOH
Remaining 4nM PhiX can be frozen and reused
?. [MiSeq Sequencing ]
[   ]  Set aside on ice until you are ready to load it onto the reagent cartridge.
?. [MiSeq Sequencing ]
[   ]  Dilute denatured PhiX library to 20 pM by adding 990 uL pre-chilled HT1 to the PhiX tube. Invert to mix.
If using a MiSeq reagent kit v2, dilute 20 pM PhiX library to 12.5 pM by adding the following volumes in a microcentrifuge tube: 375 uL 20 pM denatured PhiX library 225 uL pre-chilled HT1
?. [MiSeq Sequencing ]
[   ]  Combine library and PhiX control according to the table below:  AB1Denatured and diluted PhiX30 uL2Denatured and diluted library570 uL
AB1Denatured and diluted PhiX30 uL2Denatured and diluted library570 uL