Jul 20, 2023

Public workspaceSARS-CoV-2 Whole Genome Sequencing on Illumina V.1

DOI
dx.doi.org/10.17504/protocols.io.brfzm3p6
  • 1University College Dublin
  • UCD CFS
    Tech. support email: cfs@ucd.ie
Guerrino Macori
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DOI: dx.doi.org/10.17504/protocols.io.brfzm3p6
Protocol Citation: Guerrino Macori, Lauren Russell, Seamus Fanning 2023. SARS-CoV-2 Whole Genome Sequencing on Illumina . protocols.io https://dx.doi.org/10.17504/protocols.io.brfzm3p6Version created by Lauren Russell
Manuscript citation:
https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421
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: January 14, 2021
Last Modified: July 20, 2023
Protocol Integer ID: 46297
Keywords: Tiling PCR, WGS, SARS-CoV-2, nCoV-2019, nCoV19, WvGS
Disclaimer
In development
We are still developing and optimizing this protocol. Comments and feedback appreciated.
Abstract
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.
The SOP is adapted from the nCoV-2019 sequencing protocol: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w, and it was used in this study:
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.
https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1433/5909421
Materials
MATERIALS
ReagentNEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
ReagentDeoxynucleotide Solution Mix - 40 umol of eachNew England BiolabsCatalog #N0447L
ReagentAgencourt AMPure XP SPRI beadsBeckman CoulterCatalog #A63881
ReagentNEBNext Ultra II FS DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7805L
ReagentRandom primer mixNew England BiolabsCatalog #S1330S
ReagentSuperScript™ IV Reverse TranscriptaseThermo Fisher ScientificCatalog #18090050
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo FisherCatalog #10777019
ReagentMiSeq Reagent Nano Kit v2 (500 cycles)Illumina, Inc.Catalog #MS-103-1003
ReagentDeoxynucleotide Solution Mix - 40 umol of eachNew England BiolabsCatalog #N0447L
ReagentDTT, 100mM (Dithiothreitol)PromegaCatalog #P1171
ReagentARTIC v3 Primer PoolsIDT Technologies
ReagentNuclease-free Water - 100 mlNew England BiolabsCatalog #B1500L
ReagentTE Buffer (1X)New England BiolabsCatalog #E7808
ReagentNEBNext End Repair Module - 100 rxnsNew England BiolabsCatalog #E6050L
ReagentSodium Hydroxide NaOH 1MGibco, ThermoFisherCatalog #A4782601
ReagentPhiX Control v3Illumina, Inc.Catalog #FC-110-3001
ReagentMiSeq Reagent Kit V2 (300-cycles)illuminaCatalog #MS-102-2002


Protocol materials
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
ReagentMiSeq Reagent Nano Kit v2 (500 cycles)Illumina, Inc.Catalog #MS-103-1003
ReagentNuclease-free Water - 100 mlNew England BiolabsCatalog #B1500L
ReagentTE Buffer (1X)New England BiolabsCatalog #E7808
ReagentPhiX Control v3Illumina, Inc.Catalog #FC-110-3001
ReagentRandom primer mixNew England BiolabsCatalog #S1330S
ReagentDeoxynucleotide Solution Mix - 40 umol of eachNew England BiolabsCatalog #N0447L
ReagentAgencourt AMPure XP SPRI beadsBeckman CoulterCatalog #A63881
ReagentDTT, 100mM (Dithiothreitol)PromegaCatalog #P1171
ReagentARTIC v3 Primer PoolsIDT Technologies
ReagentSodium Hydroxide NaOH 1MGibco - Thermo Fisher ScientificCatalog #A4782601
ReagentMiSeq Reagent Kit V2 (300-cycles)Illumina, Inc.Catalog #MS-102-2002
ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo FisherCatalog #10777019
ReagentDeoxynucleotide Solution Mix - 40 umol of eachNew England BiolabsCatalog #N0447L
ReagentNEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
ReagentNEBNext End Repair Module - 100 rxnsNew England BiolabsCatalog #E6050L
ReagentNEBNext Ultra II FS DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7805L
ReagentSuperScript™ IV Reverse TranscriptaseThermo Fisher ScientificCatalog #18090050
ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
ReagentMicroAmp™ Reaction Tube with Cap, 0.2 mLThermo FisherCatalog #N8011540
ReagentRandom Primer Mix-6 nmolNew England BiolabsCatalog #S1330S
ReagentRNaseOUT Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
ReagentSuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090050
Reagenttwin.tec PCR Plate 96 LoBind semi-shirted clear 25 pcs.EppendorfCatalog #30129504
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
ReagentQubit dsDNA HS Assay KitInvitrogenCatalog #Q32851
SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
SARS-CoV-2 WvGS protocol - cDNA Preparation Reverse Transcription
2h
2h
cDNA/Reverse Transcription Section Date/Initials:_________________



In this section, the nucleic acid is extracted and used for the qPCR diagnostic test as starting material for sequencing.


[ ] In a PCR hood, mix the following reagents in a Amount0.2 mL PCR tube or PCR plate:
ABC
ReagentVolume (µL)MM for N+2 samples
60 µM random hexamers1.0
10 mM dNTPs mix (10 mM each)1.0
Template RNA 11.0
Total13.0
Master mix calculations

Note
Mastermix should be made up in the mastermix cabinet and aliquoted into PCR tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
Each reaction should have Amount13 µL when mixed.

If using master mix, it is recommended to add the Amount2 µL of the master mix to the PCR tube/plate first, then add the 11 µl of RNA to help prevent contamination.


ReagentRandom Primer Mix-6 nmolNew England BiolabsCatalog #S1330S
Lot# _______________ Exp. Date _______________

ReagentDeoxynucleotide Solution Mix - 8 umol of eachNew England BiolabsCatalog #N0447S
Lot# _______________ Exp. Date _______________

ReagentMicroAmp™ Reaction Tube with Cap, 0.2 mLThermo FisherCatalog #N8011540

[ ] Mix gently and briefly centrifuge to spin down the components, and return TemperatureOn ice .


[ ] Preheat Thermocycler to Temperature65 °C , with heated lid at Temperature105 °C


[ ] Incubate the reaction at Temperature65 °C for Duration00:05:00 , followed by an immediate snap-cool TemperatureOn ice for at least Duration00:01:00 .

[ ] In a clean Amount1.5 mL LoBind tube (96 well plates can also be used), mix together the following reagents:

ReagentVolume (uL)MM for N+2 samples
SuperScript IV RT 5X Buffer4.0
100mM DTT 1.0
RNaseOUT RNase Inhibitor1.0
Superscript IV Reverse Transcriptase1.0
Total7.0
Master mix for RT reaction.

Note
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.

ReagentRNaseOUT Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
Lot# _______________ Exp. Date _______________

ReagentSuperScript™ IV Reverse TranscriptaseThermo FisherCatalog #18090050
Lot# _______________ Exp. Date _______________

Reagenttwin.tec PCR Plate 96 LoBind semi-shirted clear 25 pcs.EppendorfCatalog #30129504

[ ] Add the above mastermix (Amount7 µL ) to the annealed DNA (Amount13 µL )) giving a total volume Amount20 µL

[ ] Cap the tube (or seal the plate), mix and then briefly centrifuge the contents.
[ ] Preheat thermocycler to Temperature42 °C , with heated lid at Temperature105 °C
[ ] Incubate sample using the following reverse transcription program:

StepTemperature (°C)TimeCycle
Reverse Transcription4250:001
RT Inactivation7010:001
Cool4HoldHold
SARS-CoV-2 Reverse Transcription Program

*PAUSE POINT cDNA can be stored at Temperature4 °C (same day) or Temperature-20 °C (up to a week).*

SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
SARS-CoV-2 WvGS protocol - ARTIC protocol - Tiled PCR
1h
1h
Tiled PCR Section Date/Initials:_________________

This section outlines the process for the tiled PCR approach from the ARTIC protocol.


Note
Primer pool sequences (v3) can be found here:
https://github.com/joshquick/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv

If required, resuspend lyophilised primers at a concentration of 100 µM each.
Prepare the primer working solution diluting to Concentration10 micromolar (µM) using Concentration0.1 % volume TE buffer.
[ ] Set up two individual reactions using primer pool 1 (set 1) and primer pool 2 (set 2) in Amount0.2 mL PCR tubes according to the following table:


ABCDE
ReagentPool 1 (uL)MM for N+2 samplesPool 2 (uL)MM for N+2 samples
Q5 Hot Start HiFi 2x MM12.512.5
Primer pool at 10uM (1 or 2)3.73.7
Nuclease-free water6.36.3
Total22.522.5
Master Mix for Tiled PCR



ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
Lot# _______________ Exp. Date _______________

[ ] Aliquot Amount22.5 µL from the mastermix into 2 96-well PCR plates or 2 sets of PCR tubes.
[ ] Add Amount2.5 µL of sample cDNA (from step 1.9) to each pool giving a total volume Amount25 µL and mix by pipetting. Spin briefly.
[ ] 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.
ABCD
StepTemperatureTimeCycles
Initial Denaturation98°C0:301
Denaturation98°C0:1535
Anneal and Extension63°C5:0035
Cool4°CHoldHold
SARS-CoV-2 Tiled PCR Program

Note
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35

Note
Pause point, Amplified cDNA can be stored at Temperature4 °C (overnight) or Temperature-20 °C up to a week.


3h 30m
SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
SARS-CoV-2 WvGS protocol - ARTIC protocol - PCR Clean-Up and Size Selection
Section for Clean-Up and Size Selection Date/Initials:_________________

Reagent preparation:
  • Allow AMPure XP beads to equilibrate to room temperature (~30 minutes). Ensure solution is homogenous prior to use, mixing gently by inversion.

Note
AMPure XP beads are needed in later steps. As the beads will need to equilibrate to room temperature before use, please consult steps 5.6 and 7.1 to ensure enough beads to cover all steps and save time.

IMPORTANT: At all stages, ensure to homogenise beads before use.


Reagent Ampure XP beads Beckman CoulterCatalog #A63881
  • Prepare the Concentration80 % volume ethanol (EtOH) using the following calculation:

Amount0.360 mL x (# Sample + 1: ________________) = ________ mL total volume (EtOH 100%)


mL total volume x 0.8 = ________ mL EtOH


Total volume _______mL - _______mL EtOH = ________mL H2O

[ ] Combine the entire volumes of pool 1 and pool 2 PCR reactions (Amount50 µL in total) into one clean PCR plate (or PCR tubes set).
[ ] Add 0.8X volume of SPRI beads per sample (Amount40 µL SPRI : Amount50 µL amplified cDNA), mix well by pipetting.
Incubate Duration00:10:00 at TemperatureRoom temperature .
[ ] Transfer the plate on the magnet and incubate for Duration00:05:00 at TemperatureRoom temperature .
[ ] Keep the plate on the magnet and remove the superanatant by pipetting from the bottom.

Note
Keep the supernatant in case you have to go back for quality assessment. You may recycle one of the PCR plates used during the pool 1/pool 2 PCR stage to retain supernatant.

Ensure to label plate correctly with step no. 3.4 and any unique identifiers for ease of finding later on.

[ ] Wash the beads in the magnet with Amount180 µL of freshly prepared 80 % volume EtOH without disturbing the pellet and incubate for Duration00:00:30 and remove the EtOH.
[ ] Repeat previous step (total 2 washes).
[ ] Spin down and place the tubes back on the magnet. Pipette off any residual ethanol with a P10 pipette and allow to dry for approximately Duration00:10:00 .
Note
Do not over-dry the beads. This may result in a lower recovery of DNA. Beads should appear dark brown and glossy. If they have become light brown or start to crack, this may be a sign they have become too dry.
*Dry beads may result in a lower recovery of DNA*

[ ] Remove the plate from the magnet and add Amount30 µL of nuclease-free water, resuspend the beads pipetting up and down at least 10 times or vortex at Centrifigation1800 rpm for Duration00:01:00
[ ] Incubate at room temperature for Duration00:02:00

[ ] Transfer the plate on the magnet and incubate for Duration00:05:00 at TemperatureRoom temperature
[ ] Carefully transfer the supernatant (28μl) into a new plate, taking care not to disturb the bead pellet.


Note
PAUSE POINT
Purified amplified cDNA can be stored at -20°C for several weeks prior to library preparation.

[ ] Quantify the sample on Qubit fluorometer or similar instrument and store completed PCR amplified cDNA prep at Temperature-20 °C
Note
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.

ReagentQubit dsDNA HS Assay KitInvitrogenCatalog #Q32851


NEBNext library preparation protocol - Fragmentation/End prep
NEBNext library preparation protocol - Fragmentation/End prep

Note
At this point in the protocol, there are two options, enzymatic fragmentation and end repair. The method used is dependant upon preference and equipment/consumable/budgetary constraints in the lab.

The enzymatic fragmentation (using NEBNext FS Library Prep Kit E7658) generates library inserts in the 150bp range compatible with 2 x 75 sequencing on illumina instruments. Follow steps 4.1 to 4.3 for this method.

The end repaire method (using NEBNext Library prep kit E7650) repairs the ends of the ~400bp amplicons generated in the tiling PCR. These libraries will be ~400bp, compatible with 2 x 250 sequencing. Follow steps 4.4 to






This section is an adaptation protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥ Amount100 ng

Note
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.



[ ] Prepare enzyme Master Mix using the following table:

ABC
ReagentVolume (uL)*(#samples+2)
NEBNext Ultra II FS Reaction Buffer 3.5 µl
NEBNext Ultra II FS Enzyme Mix 1 µl
Total Volume4.5 µl

Note
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.

[ ] Add Amount4.5 µL of prepared mastermix (above) to each well. Add Amount13 µL of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.

[ ] In a Thermocycler, with the heated lid set to 75°C, run the following program:
ABC
StepTempTime
137°C30 min
265°C30 min
Hold4°CHold


Note
OPTIMIZATION
Fragmentation 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.

ABC
Fragmentation sizeIncubation at 37°COptimization
 100 bp-250 bp 30 min 30-40 min
 150 bp-350 bp 20 min 20-30 min
 200 bp-450 bp 15 min 15-20 min
 300 bp-700 bp 10 min 5-15 min
 500 bp-1 kb 5 min 5-10 min
AB
NEBNext End Prep Vol/PCR RXN (µl)
NEBNext Ultra II End Prep Enzyme Mix 1.2
NEBNext Ultra II End Prep Reaction Buffer2.8
Total 4

AB
TemperatureTime
20°C30 minutes
65°C30 minutes
4°C∞


https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgttjwnn?step=26

Note
If necessary, samples can be stored at –Temperature-20 °C , however, a slight loss in yield (~20%) may be observed. It is recommend continuing with adaptor ligation before stopping.

Continue with this protocol from step 5.



Steps 4.1 to 4.3 detailed enzymatic fragmentation. The following steps (4.4 to 4.6) detail the end repair option.

*If you have carried out steps 4.1 to 4.3, this protocol continues from step 5*


[ ] Prepare the following mastermix in a sterile nuclease-free tube:


AB
ComponentVolume
NEBNext Ultra II End Prep Enzyme Mix1.5 µl
NEBNext Ultra II End Prep Reaction Buffer3.5 µl
Total Volume5 µl


[ ] Add Amount5 µL of mastermix (above) to each well. Add Amount25 µL of purified DNA to the PCR tube or to the wells of the PCR plate.Vortex the reaction for 5 seconds and briefly spin in a microcentrifuge.



[ ] In a thermocycler, with the heated lid set to Temperature75 °C , run the following program:

AB
TemperatureTime
20 °C30 min
65 °C30 min
4 °C∞


Note
If necessary, samples can be stored at –Temperature-20 °C , 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
NEBNext library preparation protocol - Adapter ligation
[ ] Add the following components directly to the FS Reaction Mixture:

AB
ComponentVolume
FS Reaction Mixture (Step 4.3) or End Prep Reaction Mixture (step 4.6)17.5 µl/ 30 µl
NEBNext Ultra II Ligation Master Mix 15 µl
NEBNext Adaptor for Illumina 1.25µl
Total Volume33.75 µl/ 46.25 µl

Note
It is not recommended to add adaptor to a premix in the Adaptor Ligation Step.

For this reason, add Ligation Master mix to each well/PCR tube, then add 1.25μl adapter at the end. Seal plate, vortex for Duration00:00:10 & spin briefly


[ ] Incubate at Temperature20 °C for Duration00:15:00 in a thermocycler with the heated lid off.
[ ] Add Amount1.5 µL µl of USER Enzyme to the ligation mixture from Step 5.1. Vortex for Duration00:00:10 & spin briefly.

10s
[ ] Mix well and incubate in thermocycler at Temperature37 °C for Duration00:15:00 with the heated lid set to ≥ Temperature47 °C

Note
Samples can be stored overnight at Temperature-20 °C


Cleanup of Adaptor-ligated DNA
Cleanup of Adaptor-ligated DNA


Note
The volumes of Ampure XP Breads will vary depend on fragmentation method used in section 4.



[ ] Vortex SPRIselect or NEBNext Sample Purification Beads to resuspend.
[ ] Add Amount28 µL (FS fragmentation) or Amount43 µL (end repair) of the Ampure XP Beads to the ligation reaction mixture and mix well by pipetting up and down, or vortex. Spin briefly.

[ ] Incubate at room temperature for Duration00:05:00
[ ] Place the plate on magnetic block for Duration00:05:00
[ ] Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets




[ ] Wash the beads adding Amount200 µL of freshly prepared 80% ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for Duration00:00:30 , and then carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets.
[ ] Repeat Step 5.10 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.
[ ] Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.

Note
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.

[ ] Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding Amount10 µL Concentration0.1 % volume TE (dilute 1X TE Buffer 1:10 in water).

[ ] Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least Duration00:02:00 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.

[ ] Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer Amount7.5 µL to a new PCR tube.

Note
Samples can be stored at Temperature-20 °C


NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA
NEBNext library preparation protocol - PCR Enrichment of Adapter-ligated DNA
[ ] Add the following reagents to each well from step 5.15

AB
ComponentVolume
Adaptor Ligated DNA Fragments (Step 5.15)7.5 µl
NEBNext Ultra II Q5 Master Mix 12.5 µl
Index Primer/i7 Primer 2.5 µl
Universal PCR Primer/i5 Primer 2.5 µl
Total Volume 25 µl

Note
Ensure to take note of what index set (1 or 2) is used and their sequence numbers,

Index set no. _______________

Index Range (A) _______________ Index Range (B) _______________
[ ] 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.
[ ] Place the tube/plate on a thermocycler with the heated lid set to Temperature105 °C and perform PCR amplification using the following PCR cycling conditions:



CYCLE STEPTEMPTIMECYCLES
Initial Denaturation98°C30 seconds1
Denaturation98°C10 seconds5*
Annealing/Extension65°C75 seconds
Final Extension65°C5 minutes1
Hold4°C∞

Note
*Cycle number was determined by size of input DNA ~100ng is 4-5 cycles.

NEBNext library preparation protocol - Clean up of PCR reaction
NEBNext library preparation protocol - Clean up of PCR reaction
Allow the Ampure XP beads to warm to room temperature for at least 30 minutes before use.

[ ] Vortex SPRIselect to resuspend.
[ ] Add Amount22.5 µL (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.
[ ] Incubate samples on bench top for at least Duration00:05:00 at TemperatureRoom temperature

[ ] 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.
[ ] After Duration00:05:00 (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).

[ ] Add Amount200 µL of Concentration80 % volume freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for Duration00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
[ ] Repeat Step 7.5. 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.
[ ] Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Note
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.

[ ] Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding Amount17 µL of Concentration0.1 % (v/v) TE (dilute 1X TE Buffer 1:10 in water).

[ ] Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least Duration00:02:00 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.

[ ] Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer Amount15 µL to a new PCR tube and store at Temperature-20 °C .

Assess Library quality
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.

Note
In this protocol Amount2 µL of library (Amount198 µL buffer)


[ ] Run Samples on Agilent Bioanalyser or Agilent Tapestation to 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.
Note
Tape station D1000 HS Amount2 µL of library in Amount2 µL buffer (ladder Amount2 µL in Amount2 µL buffer for each cartridge)


[ ] Calculate the dilutions required to normalise each sample to a 4nM concentration using the following formula:

Note
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.

[ ] Run Samples on a bioanalyser or tapestation 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
Note
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
MiSeq Sequencing
6m
6m
Pooling and Library Denaturation Date/Initials:_________________


This section demonstrates how to generate a pooled library for V2 reagents on the MiSeq.
Note
Thaw the MiSeq reagents overnight or in aTemperatureRoom temperature waterbath.
Remove HT1 from freezer and thaw at TemperatureRoom temperature .
Store at Temperature2 °C toTemperature8 °C until you are ready to dilute denatured libraries.


Note
Label 3 eppendorfs for:
(1) the pooled library
(2) denaturation of library
(3) 0.2N NaOH


Make a fresh dilution of 0.2N of NaOH by combining the following volumes in a microcentrifuge tube: Amount800 µL laboratory-grade water and Amount200 µL stock Concentration1.0 nanomolar (nM) NaOH


[ ] Pool Amount5 µL of each normalised sample into an eppendorf tube. This will be (1) pooled library.
[ ] Combine the following volumes in a microcentrifuge tube (2):
Amount5 µL 4nM pooled library and Amount5 µL of 0.2 N NaOH.

[ ] Vortex briefly and then centrifuge at 280 x g for 1 minute.
[ ] Incubate at room temperature for Duration00:05:00
[ ] Add Amount990 µL of pre-chilled HT1 to the tube containing the denatured library (2). The result is 1 mL of a 20 pM denatured library.
[ ] Dilute the 20 pM library to the desired concentration, see table below:

Concentration6 pM8 pM10 pM12 pM15 pM20 pM
20 pM library180 uL240 uL300 uL360 uL450 uL600 uL
Pre-chilled HT1420 uL360 uL300 uL240 uL150 uL0 uL

Note
We recommend diluting the library to 10pM for optimal cluster density during Miseq runs with V2 reagents.


[ ] Invert to mix and then pulse centrifuge




Note
The following steps 10.5 to 10.7 can be carried out ahead of time and PhiX library can be stored at Temperature-20 °C for a number of weeks

[ ] Dilute stock PhiX to 4nM by combining:
- Amount2 µL of Concentration10 nanomolar (nM) PhiX library
- Amount3 µL of Concentration10 millimolar (mM) Tris-Cl, pH 8.5 with 0.1% Tween 20

Denature the PhiX control by adding the following volumes in a microcentrifuge tube:
- Amount5 µL of Concentration4 nanomolar (nM) PhiX library
- Amount5 µL of Concentration0.2 nanomolar (nM) NaOH

Note
Remaining Concentration4 nanomolar (nM) PhiX can be frozen and reused


[ ] Vortex briefly to mix and centrifuge at Shaker280 x g forDuration00:01:00 .
[ ] Incubate atTemperatureRoom temperature for Duration00:05:00



6m
[ ] Dilute denatured PhiX library to 20 pM by adding 990 uL pre-chilled HT1 to the PhiX tube. Invert to mix.

Note
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 µL 20 pM denatured PhiX library
- 225 µL pre-chilled HT1

[ ] Combine library and PhiX control according to the table below:

AB
Denatured and diluted PhiX (12.5pM)30 µl
Denatured and diluted library (10 pM)570 µl



[ ] Set aside on ice until you are ready to load it onto the reagent cartridge.
[ ] Mix reagents of the MiSeq cartridge thoroughly by inverting several times.

[ ] Using a fresh 1000 µL pipette tip, transfer the denatured and library (with PhiX spiked) into position 17.

[ ] Load the sample sheet and reagents according to onscreen instructions in the MiSeq Control software.