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

[DESCRIPTION]
This protocol is an adaption of several circulating protocols on SARS-CoV-2 sequencing using the ARTIC protocol and the Illumina DNA Prep library kit.  Its purpose is to simplify things for the average state public health laboratory, using equipment and expertise they currently possess, most likely from their funded PulseNet activities.

Feedback and comments appreciated.

[STEPS]
SECTION: DNA Prep - Before you begin
1. 
Before You Begin

This protocol is an adaptation of the Illumina DNA Prep kit (below).  It is laid out specifically for using amplicons from the ARTIC Tiling PCR 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 link for the Illumina DNA Prep reagents. The consumables and equipment document (CED) should 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 Illumina DNA Prep, (M) Tagmentation kit reagents are not listed in the consumables document so they have been added to Table 1 below.

For Sample Indices please pay attention to which version you are using as there are several different formats requiring different steps. This protocol is written for 96 Dual Index plate format indices.

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.

Illumina DNA Prep Reference Guide

Illumina DNA Prep Checklist

Illumina DNA Prep Consumable and Equipment (.zip file)

Index Adapters Pooling Guide

  
  
 
 

All reagents except EPM should be brought to room temperature prior to use. EPM should be kept on ice. Check TSB for precipitates. If there are any, heat at 37 °C for 10 min , then vortex until dissolved.

SECTION: DNA Prep - Dilution Plate Preparation
2. Dilution Plate Preparation        Date/Initials:_________________

Prior to starting your DNA 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 30 µL final volume of sample contains 100 ng to 500 ng of DNA.  Less than 100 ng 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.



SECTION: DNA Prep - Dilution Plate Preparation
2.1. [   ]  Label a 96 well dilution plate with the number of columns required.


SECTION: DNA Prep - Dilution Plate Preparation
2.2. [   ]  Add enough DNA to reach a total of at least 100 ng** and add molecular grade water (CED) to bring the total volume to 30 µL.

 


SECTION: DNA Prep - Tagmentation
3. Tagmentation        Date/Initials:_________________

This step uses the Bead-Linked Transposomes (BLT) to tagment DNA, which is a process that fragments and
tags the DNA with adapter sequences.



SECTION: DNA Prep - Tagmentation
3.1. [   ]  Prepare tagmenation Master Mix using Calculation 1:
 
 

 

SECTION: DNA Prep - Tagmentation
3.2. [   ]  Vortex master mix, and then add 20 µL to each sample well. Pipette each 10 times to mix.
SECTION: DNA Prep - Tagmentation
3.3. [   ]  Cover and seal the plate with Microseal 'B' (CED).
SECTION: DNA Prep - Tagmentation
3.4. [   ]  Place the plate in the thermal cycler and run the TAG Program below: 55°C

 


SECTION: DNA Prep - Post Tagmentation Clean-up
4. 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 Room temperature 

SECTION: DNA Prep - Post Tagmentation Clean-up
4.1. [   ]  Check TSB for precipitates, and then add 10 µL to each tagmentation reaction (sample well). Slowly pipette to mix 10 times to resuspend the beads.


 

SECTION: DNA Prep - Post Tagmentation Clean-up
4.2. [   ]  Seal the plate with Microseal 'B' (CED), place on the preprogrammed thermal cycler, and run the PTC
Program.

 

SECTION: DNA Prep - Post Tagmentation Clean-up
4.3. [   ]  Place plate on magnet 3 min or until clear.
SECTION: DNA Prep - Post Tagmentation Clean-up
4.4. [   ]  Remove and discard supernatant.
SECTION: DNA Prep - Post Tagmentation Clean-up
4.5. [   ]  Remove from magnet, and add 100 µL TWB. Pipette to mix**.

 

SECTION: DNA Prep - Post Tagmentation Clean-up
4.6. [   ]  Place back on magnet 3 min or until clear. Remove and discard supernatant.
SECTION: DNA Prep - Post Tagmentation Clean-up
4.7. [   ]  Repeat TWB washes described above once more, then slowly add 100 µL  TWB to the beads. Pipette to mix.

SECTION: DNA Prep - Amplify Tagmented DNA
5. 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 the
indexes selected for low plexity pooling have the appropriate color balance, see the Index Adapters Pooling Guide.
SECTION: DNA Prep - Amplify Tagmented DNA
5.1. [   ]  Prepare PCR master mix using Calculation 2:
 
 

 
SECTION: DNA Prep - Amplify Tagmented DNA
5.2. [   ]  Vortex and quick spin master mix.
SECTION: DNA Prep - Amplify Tagmented DNA
5.3. [   ]  Remove and discard TWB from samples. Remove from magnet, and immediately add 40 µL PCR master mix, pipette to mix until beads are fully resuspended, then quick spin. 

SECTION: DNA Prep - Amplify Tagmented DNA
5.4. [   ]  Add 10 µL indices**, and then pipette to mix.

 


SECTION: DNA Prep - Amplify Tagmented DNA
5.5. [   ]  Seal the plate with Microseal 'B' (CED), place on the thermal cycler, and run the BLT PCR Program on the thermal cycler.

 

 

SECTION: DNA Prep - Clean-up Libraries
6. Clean-up Libraries        Date/Initials:_________________

This step uses double-sided bead purification procedure to purify the amplified libraries.
SECTION: DNA Prep - Clean-up Libraries
6.1. [   ]  Centrifuge plate at 280 x g, 1 min , and then place on magnet for 5 min or until clear

SECTION: DNA Prep - Clean-up Libraries
6.2. [   ]  Transfer 45 µL of supernatant to clean wells of a new midi plate (CED), and then remove from magnet

SECTION: DNA Prep - Clean-up Libraries
6.3. [   ]  Vortex and invert SPB until completely resuspended.   
SECTION: DNA Prep - Clean-up Libraries
6.4. [   ]  Add 81 µLof SPB** to samples, pipette 10 times to mix.
 

SECTION: DNA Prep - Clean-up Libraries
6.5. [   ]  Seal (CED) plate and incubate for at least 5 min.


SECTION: DNA Prep - Clean-up Libraries
6.6. [   ]  Prepare fresh 80 % volume EtOH using the following calculation:

0.4 mL * (#samples+1) = _______ mL volume EtOH
0.1 mL * (#samples+1) = _______mL volume molecular grade water

Add those two volumes together for 80 % volume EtOH.
SECTION: DNA Prep - Clean-up Libraries
6.7. [   ]  Place on magnet for 5 min or until clear. Remove and discard supernatant.
SECTION: DNA Prep - Clean-up Libraries
6.8. [   ]  Perform two 30 s washes with 200 µL 80 % volume 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.
SECTION: DNA Prep - Clean-up Libraries
6.9. [   ]  Air dry beads approx. 5 min  or until pellet no longer appears shiny and has one or two cracks in it and then remove plate from magnet


 

SECTION: DNA Prep - Clean-up Libraries
6.10. [   ]  Vortex RSB and add 32 µL. Pipette to mix and incubate at Room temperature for 2 min .

SECTION: DNA Prep - Clean-up Libraries
6.11. [   ]  Place plate on magnet for 2 min. Transfer 30 µL supernatant to new wells. 

SECTION: DNA Prep - Clean-up Libraries
6.12. [   ]  Library is now ready for quantification. 

 

SECTION: DNA Prep - Qubit Quantification
7. Qubit Quantification        Date/Initials:_________________

It is also recommended to check library quality by fragment analysis using High Sensitivity DNA kits for Agilent TapeStation, Bioanalyzer, or other similar high throughput methodology.


SECTION: DNA Prep - Qubit Quantification
7.1. [   ]  Prepare Qubit working solution using Calculation 5. Label assay tubes for samples and standards.

 


SECTION: DNA Prep - Qubit Quantification
7.2. [   ]  Combine 190 µL Qubit working solution + 10 µL Qubit standard into labeled standard tubes

SECTION: DNA Prep - Qubit Quantification
7.3. [   ]  Combine 198 µL Qubit working solution + 2 µL extracted DNA into labeled tubes

SECTION: DNA Prep - Qubit Quantification
7.4. [   ]  Vortex all tubes for 2-3 sec and incubate at Room temperature for minimum of 2 min. Read tubes within 60 min

SECTION: DNA Prep - Qubit Quantification
7.5. [   ]  Record sample results.
SECTION: DNA Prep - Denaturation of Pooled Library
8. Denaturation of Pooled Library        Date/Initials:_________________

This section demonstrates how to generate a pooled library for V3 reagents on the MiSeq.
SECTION: DNA Prep - Denaturation of Pooled Library
8.1. [   ]  Centrifuge plate at 280 x g, 1 min
SECTION: DNA Prep - Denaturation of Pooled Library
8.2. [   ]  Make 4 Nanomolar (nM)pool using Calculation 6 
 
     Calculation 6: 4nM Pool        Pool conc.  (Qubit value, ng/µl)               Molarity (nM)=  (Pool conc. x 1000) / 528               Volume of pool to dilute (µl)=  (4 x 50) / Molarity               Volume of RSB to dilute (µl)= (50   – Pool to dilute)          
 
SECTION: DNA Prep - Denaturation of Pooled Library
8.3. [   ]  Add 400 µL molecular grade H20 to the 100 µL 1.0 N NaOH aliquot for 0.2 N NaOH

SECTION: DNA Prep - Denaturation of Pooled Library
8.4. [   ]  Combine 5 µL 0.2 N NaOH and 5 µL pooled DNA in a 1.5 mL tube. Incubate 5 min at Room temperature

SECTION: DNA Prep - Denaturation of Pooled Library
8.5. [   ]  Immediately add 990 µL pre-chilled HT1. Pipette to mix, then use Table 2 for desired library pool concentration
 
     Desired final loading   concentration      □8pM      □9pM      □10pM      □12pM      □14pM      □15pM      □16pM      □18pM      □20pM        Denatured Pooled   Library      800µl      900µl      1000µl      600µl      700µl      750µl      800µl      900µl      1000µl        HT1      200µl      100µl      0µl      400µl      300µl      250µl      200µl      100µl      0µl   
 
SECTION: DNA Prep - Post Tagmentation Clean-up
4.8. [   ]  Place back on magnet 3 min or until clear.