TITLE: SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 2 - Illumina DNA Flex Protocol
AUTHORS: Joel Sevinsky, Arian Nassiri, Heather Blankenship, Erin Young, Kevin Libuit, Kelly Oakeson, Lauren Turner, StaPH-B Consortium

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is an adaption of several circulating protocols on SARS-CoV-2 sequencing using the ARTIC protocol and the Illumina Nexterra DNA Flex library prep kit.  Its purpose is to simplify things for the average state public health laboratory, using equipment and expertise they currently posess, most likely from their funded PulseNet activities.</div><div class = "text-block"><span style = "font-weight:bold;">Feedback and comments appreciated.</span></div></div>

[STEPS]
?. [DNA Flex - Dilution Plate Preparation]
Dilution Plate Preparation        Date/Initials:_________________Prior to starting your DNA Flex library prep, samples should be diluted into a 96 well plate as described below.  Ideally, you would like each sample to be diluted such that the  final volulme of sample contains  to  of DNA.  Less than  of DNA may cause the sample to be under represented in the pool, and modifications to increase the concentration of that sample are not practical when multiplexing the library prep.  Much of this protocol can be achieved using 96 well plates, so having specimens organized in columns allows the use of an 8 channel multi-channel pipette.
30 µl
100 ng
500 ng
100 ng
?. [DNA Flex - Dilution Plate Preparation]
[   ]  Label a 96 well dilution plate with the number of columns required.
?. [DNA Flex - Dilution Plate Preparation]
[   ]  Add enough DNA to reach a total of at least ** and add molecular grade water (CED) to bring the total volume to .
100 ng
30 µl
**NOTE: Preferred amount is   to  .  Less than that can lead to under representation of the sample in the final pool.  More is probably ok as long as it is not extreme.
?. [DNA Flex - Tagmentation]
Tagmentation        Date/Initials:_________________This step uses the Bead-Linked Transposomes (BLT) to tagment DNA, which is a process that fragments andtags the DNA with adapter sequences.
?. [DNA Flex - Tagmentation]
[   ]  Prepare tagmenation Master Mix using Calculation 1:  ABC1ReagentVolume (uL)*(#samples+2)2BLT**10.03TB1**10.0
ABC1ReagentVolume (uL)*(#samples+2)2BLT**10.03TB1**10.0
**NOTE: Vortex BLT prior to using.  Both reagents should be at room temperature before using.
?. [DNA Flex - Tagmentation]
[   ]  Vortex master mix, and then add  to each sample well. Pipette each 10 times to mix.
20 µl
?. [DNA Flex - Tagmentation]
[   ]  Place the plate in the thermal cycler and run the TAG Program below: 55°C  ABC1StepTempTime2Tagmentation55°C15 min3Hold10°CHold
ABC1StepTempTime2Tagmentation55°C15 min3Hold10°CHold
?. [DNA Flex - Post Tagmentation Clean-up]
Post Tagmentation Clean-up        Date/Initials:_________________This step stops the tagmentation and washes the adapter-tagged DNA on the BLT before PCR amplification. All reagents should be at
0 Room temperature
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Check TSB for precipitates, and then add  to each tagmentation reaction (sample well). Slowly pipette to mix 10 times to resuspend the beads.
10 µl
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Seal the plate with Microseal 'B' (CED), place on the preprogrammed thermal cycler, and run the PTCProgram.  ABC1StepTempTime2Tagemntation Stop37°C15 min3Cool10°CHold
ABC1StepTempTime2Tagemntation Stop37°C15 min3Cool10°CHold
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Place plate on magnet  or until clear.
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Remove from magnet, and add  TWB. Pipette to mix**.
100 µl
**NOTE: A deliberately slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Place back on magnet  or until clear. Remove and discard supernatant.
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Repeat TWB washes described above 2 more times. Wait to discard supernatant and leave in magnet after second wash.
?. [DNA Flex- Amplify Tagmented DNA]
Amplify Tagmented DNA        Date/Initials:_________________This step amplifies the tagmented DNA using a limited-cycle PCR program. The PCR step adds Index 1 (i7)adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. To confirm theindexes selected for low plexity pooling have the appropriate color balance, see the Index Adapters Pooling Guide.
?. [DNA Flex- Amplify Tagmented DNA]
[   ]  Prepare PCR master mix using Calculation 2:  ABC1ReagentVolume (uL)*(#samples+2)2EPM20.03H2O20.0
ABC1ReagentVolume (uL)*(#samples+2)2EPM20.03H2O20.0
?. [DNA Flex- Amplify Tagmented DNA]
[   ]  Vortex and quick spin master mix.
?. [DNA Flex- Amplify Tagmented DNA]
[   ]  Remove and discard TWB from samples. Remove from magnet, and immediately add  PCR master mix, pipette to mix.
40 µl
?. [DNA Flex- Amplify Tagmented DNA]
[   ]  Add  indices**, and then pipette to mix.
10 µl
**NOTE: Use the same index strategy that you use for PusleNet organisms. If you are unfamiliar with the PulseNet protocol, see Index Adapters Pooling Guide for guidance on the indexing. A separate Appendix will be created discussing pooling best practices.
?. [DNA Flex- Amplify Tagmented DNA]
[   ]  Seal the plate with Microseal 'B' (CED), place on the thermal cycler, and run the BLT PCR Program on the thermal cycler.  ABCD1StepTempTimeCycles2Elute68°C03:0013Denature98°C03:0014Denature98°C00:455**5Anneal62°C00:305**6Extend68°C02:005**7Extend68°C01:0018Hold10°CHold1
ABCD1StepTempTimeCycles2Elute68°C03:0013Denature98°C03:0014Denature98°C00:455**5Anneal62°C00:305**6Extend68°C02:005**7Extend68°C01:0018Hold10°CHold1
**NOTE: This is a 5 cycle PCR reaction designed for the majority of samples in 96 well plate sequencing. If your samples started with less than 100 ng of DNA, or eventually failed for low coverage in your pool, refer to page 10 of the Nextera DNA Flex Library Prep Reference Guide for guidelines on how to increase the number of cycles for low input samples.
?. [DNA Flex - Clean-up Libraries]
Clean-up Libraries        Date/Initials:_________________This step uses double-sided bead purification procedure to purify the amplified libraries.
?. [DNA Flex - Clean-up Libraries]
[   ]  Centrifuge plate at  , and then place on magnet for  or until clear
Centrifuge: 280 34, 1 min
?. [DNA Flex - Clean-up Libraries]
[   ]  Transfer  of supernatant to clean wells of a new midi plate (CED), and then remove from magnet
45 µl
?. [DNA Flex - Clean-up Libraries]
[   ]  Prepare fresh  EtOH using the following calculation: * (#samples+1) = _______ mL volume EtOH * (#samples+1) = _______mL volume molecular grade waterAdd those two volumes together for  EtOH.
0.4 ml
0.1 ml
?. [DNA Flex - Clean-up Libraries]
[   ]  Seal (CED) plate and incubate for at least .
?. [DNA Flex - Clean-up Libraries]
[   ]  Vortex and invert SPB.
?. [DNA Flex - Clean-up Libraries]
[   ]  Library is now ready for quantification.
SAFE STOPPING POINTIf you are stopping, seal the plate with Microseal 'B' adhesive or Microseal 'F' foil seal, and store at-25°C to -15°C for up to 30 days.
?. [DNA Flex - Clean-up Libraries]
[   ]  Place plate on magnet for . Transfer  supernatant to new wells.
30 µl
?. [DNA Flex - Clean-up Libraries]
[   ]  Vortex RSB and add . Pipette to mix and incubate at  for  .
32 µl
0 Room temperature
?. [DNA Flex - Clean-up Libraries]
[   ]  Air dry beads approx.  and then remove plate from magnet
?. [DNA Flex - Clean-up Libraries]
[   ]  Perform two  washes with  EtOH. Remove and discard supernatant after each wash. After second wash make sure to remove residual EtOH with 20 uL pipette. Perform this step on the magnetic stand without disturngin the beads.
200 µl
?. [DNA Flex - Clean-up Libraries]
[   ]  Place on magnet for  or until clear. Remove and discard supernatant.
?. [DNA Flex - Qubit Quantification]
Qubit Quantification        Date/Initials:_________________
?. [DNA Flex - Qubit Quantification]
[   ]  Prepare Qubit working solution using Calculation 5. Label assay tubes for samples and standards.  ABCDE1ReagentVolume (uL)*(#rxns+2std+2)Lot#Exp. Date2Qubit Reagent1.03Qubit Buffer199.04Total200.0
ABCDE1ReagentVolume (uL)*(#rxns+2std+2)Lot#Exp. Date2Qubit Reagent1.03Qubit Buffer199.04Total200.0
?. [DNA Flex - Qubit Quantification]
[   ]  Combine  Qubit working solution +  Qubit standard into labeled standard tubes
190 µl
10 µl
?. [DNA Flex - Qubit Quantification]
[   ]  Combine  Qubit working solution +  extracted DNA into labeled tubes
198 µl
2 µl
?. [DNA Flex - Qubit Quantification]
[   ]  Vortex all tubes for 2-3 sec and incubate at  for minimum of . Read tubes within
0 Room temperature
?. [DNA Flex - Qubit Quantification]
[   ]  Record sample results.
?. [DNA Flex - Denaturation of Pooled Library]
Denaturation of Pooled Library        Date/Initials:_________________This section demonstrates how to generate a pooled library for V3 reagents on the MiSeq.
?. [DNA Flex - Denaturation of Pooled Library]
[   ]  Centrifuge plate at
Centrifuge: 280 34, 1 min
?. [DNA Flex - Denaturation of Pooled Library]
[   ]  Make pool using Calculation 6   AB1Pool conc.  (Qubit value, ng/µl)     2Molarity (nM)=  (Pool conc. x 1000) / 528     3Volume of pool to dilute (µl)=  (4 x 50) / Molarity     4Volume of RSB to dilute (µl)= (50 – Pool to dilute)
AB1Pool conc.  (Qubit value, ng/µl)     2Molarity (nM)=  (Pool conc. x 1000) / 528     3Volume of pool to dilute (µl)=  (4 x 50) / Molarity     4Volume of RSB to dilute (µl)= (50 – Pool to dilute)
?. [DNA Flex - Denaturation of Pooled Library]
[   ]  Add  molecular grade H20 to the  1.0 N NaOH aliquot for 0.2 N NaOH
400 µl
100 µl
?. [DNA Flex - Denaturation of Pooled Library]
[   ]  Combine  0.2 N NaOH and  pooled DNA in a  tube. Incubate  at
5 µl
5 µl
1.5 ml
0 Room temperature
?. [DNA Flex - Denaturation of Pooled Library]
[   ]  Immediately add  pre-chilled HT1. Pipette to mix, then use Table 2 for desired library pool concentration  ABCDEFGHI1Final Library Conc (pM)8101214151618202Pooled Library (uL)400.0500.0600.0700.0750.0800.0900.01000.03HT1 (uL)600.0500.0400.0300.0250.0200.0100.00.0
990 µl
ABCDEFGHI1Final Library Conc (pM)8101214151618202Pooled Library (uL)400.0500.0600.0700.0750.0800.0900.01000.03HT1 (uL)600.0500.0400.0300.0250.0200.0100.00.0
?. [DNA Flex - Before you begin]
Before You BeginThis protocol is an adaptation of the Illumina Nextera DNA Flex Library Prep kit (below).  It is laid out specifically for using amplicons from the ARTIC Protocol.  The audience is assumed to be public health laboratorians with access to instruments and reagents used for PulseNet WGS. Four documents are linked below for reference, and can also be found following the reagent link for the Nextera DENA Flex Library Prep. The consumables and equipment document (CED) shoud be used to make sure you have the appropriate materials before beginning. There are too many consumables to list out individually in this protocol so please use the document and fill out your Lot# and Exp Dates on this document as well. The Nextera DNA Flex Library Prep kit reagents are not listed in the consumables document so they have been added to Table 1 below.Your lab will most likely have everything already for their PulseNet activities.  The exception to this is PhiX which will be listed in the appropriate step.Nextera DNA Flex Library Prep Reference GuideNextera DNA Flex Library Prep ChecklistNextera DNA Flex Library Prep Consumables and EquipmentIndex Adapters Pooling Guide  ABCDE1Box #ReagentDescriptionLot #Exp. Date21SPBSample Purification Beads31TSBTagment Stop Buffer41TWBTagment Wash Buffer52RSBResuspension Buffer62TB1Tagmentation Buffer 172EPMEnhanced PCR Mix83BLTBead-Linked Transposome
ABCDE1Box #ReagentDescriptionLot #Exp. Date21SPBSample Purification Beads31TSBTagment Stop Buffer41TWBTagment Wash Buffer52RSBResuspension Buffer62TB1Tagmentation Buffer 172EPMEnhanced PCR Mix83BLTBead-Linked Transposome
?. [DNA Flex - Post Tagmentation Clean-up]
[   ]  Remove and discard supernatant.
?. [DNA Flex - Tagmentation]
[   ]  Cover and seal the plate with Microseal 'B' (CED).
?. [DNA Flex - Clean-up Libraries]
[   ]  Add of SPB** to samples, pipette 10 times to mix.
81 µl
**NOTE: Since we are using this kit on small amplicons from the ARTIC Protocol, we will follow the DNA Flex instructions for small amplicon clean up, not standard DNA input.