Apr 29, 2020
Version 1
SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 2 - Illumina DNA Flex Protocol V.1
- Joel Sevinsky1,
- Arian Nassiri2,
- Heather Blankenship3,
- Erin Young4,
- Kevin Libuit2,
- Kelly Oakeson4,
- Lauren Turner2,
- StaPH-B Consortium5
- 1Theiagen Genomics;
- 2Virginia Division of Consolidated Laboratory Services;
- 3Michigan Department of Health and Human Services;
- 4Utah Public Health Laboratory;
- 5State Public Health Bioinformaticians
- Coronavirus Method Development Community
- StaPH-B
Protocol Citation: Joel Sevinsky, Arian Nassiri, Heather Blankenship, Erin Young, Kevin Libuit, Kelly Oakeson, Lauren Turner, StaPH-B Consortium 2020. SARS-CoV-2 Sequencing on Illumina MiSeq Using ARTIC Protocol: Part 2 - Illumina DNA Flex Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bffyjjpw
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: In development
We are still developing and optimizing this protocol. Comments and feedback appreciated.
Created: April 22, 2020
Last Modified: April 29, 2020
Protocol Integer ID: 36056
Keywords: SARS-CoV-2, ARTIC, StaPH-B,
Abstract
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.
Feedback and comments appreciated.
Materials
STEP MATERIALS
Nextera DNA Flex Library PrepIllumina, Inc.Catalog #20018705
Protocol materials
Nextera DNA Flex Library PrepIllumina, Inc.Catalog #20018705
Nextera DNA Flex Library PrepIllumina, Inc.Catalog #20018705
DNA Flex - Before you begin
DNA Flex - Before you begin
Before You Begin
This 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 PrepIllumina, Inc.Catalog #20018705
| Box # | Reagent | Description | Lot # | Exp. Date | |
| 1 | SPB | Sample Purification Beads | |||
| 1 | TSB | Tagment Stop Buffer | |||
| 1 | TWB | Tagment Wash Buffer | |||
| 2 | RSB | Resuspension Buffer | |||
| 2 | TB1 | Tagmentation Buffer 1 | |||
| 2 | EPM | Enhanced PCR Mix | |||
| 3 | BLT | Bead-Linked Transposome |
Table 1: Reagent List from Nextera DNA Flex Library Prep Kit
DNA Flex - Dilution Plate Preparation
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 30 µL final volulme 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.
[ ] Label a 96 well dilution plate with the number of columns required.
[ ] 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 .
Note
**NOTE: Preferred amount is 100 ng to 500 ng . 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
DNA Flex - Tagmentation
40m
40m
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.
40m
[ ] Prepare tagmenation Master Mix using Calculation 1:
| Reagent | Volume (uL) | *(#samples+2) | |
| BLT** | 10.0 | ||
| TB1** | 10.0 |
Calculation 1: Tagmentation Master Mix
Note
**NOTE: Vortex BLT prior to using. Both reagents should be at room temperature before using.
[ ] Vortex master mix, and then add 20 µL to each sample well. Pipette each 10 times to mix.
[ ] Cover and seal the plate with Microseal 'B' (CED).
[ ] Place the plate in the thermal cycler and run the TAG Program below: 55°C
| Step | Temp | Time | |
| Tagmentation | 55°C | 15 min | |
| Hold | 10°C | Hold |
TAG Program: Thermal cycler should be set for 50 uL volume and lid set to 105°C.
DNA Flex - Post Tagmentation Clean-up
DNA Flex - Post Tagmentation Clean-up
45m
45m
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
45m
[ ] 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.
[ ] Seal the plate with Microseal 'B' (CED), place on the preprogrammed thermal cycler, and run the PTC
Program.
| Step | Temp | Time | |
| Tagemntation Stop | 37°C | 15 min | |
| Cool | 10°C | Hold |
PTC Program: Thermal cycler should be set for 60 uL volume and lid set to 105°C.
[ ] Place plate on magnet 00:03:00 or until clear.
[ ] Remove and discard supernatant.
[ ] Remove from magnet, and add 100 µL TWB. Pipette to mix**.
Note
**NOTE: A deliberately slow pipetting technique minimizes the potential of TWB foaming to avoid incorrect volume aspiration and incomplete mixing.
[ ] Place back on magnet 00:03:00 or until clear. Remove and discard supernatant.
[ ] Repeat TWB washes described above 2 more times. Wait to discard supernatant and leave in magnet after second wash.
DNA Flex- Amplify Tagmented DNA
DNA Flex- Amplify Tagmented DNA
45m
45m
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.
45m
[ ] Prepare PCR master mix using Calculation 2:
| Reagent | Volume (uL) | *(#samples+2) | |
| EPM | 20.0 | ||
| H2O | 20.0 |
Calculation 2: PCR Master Mix
[ ] Vortex and quick spin master mix.
[ ] Remove and discard TWB from samples. Remove from magnet, and immediately add 40 µL PCR master mix, pipette to mix.
[ ] Add 10 µL indices**, and then pipette to mix.
Note
**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.
[ ] Seal the plate with Microseal 'B' (CED), place on the thermal cycler, and run the BLT PCR Program on the thermal cycler.
| Step | Temp | Time | Cycles | |
| Elute | 68°C | 03:00 | 1 | |
| Denature | 98°C | 03:00 | 1 | |
| Denature | 98°C | 00:45 | 5** | |
| Anneal | 62°C | 00:30 | 5** | |
| Extend | 68°C | 02:00 | 5** | |
| Extend | 68°C | 01:00 | 1 | |
| Hold | 10°C | Hold | 1 |
BLT PCR Program: Volume should be set for 50 uL and the lid temperature set to 105°C.
Note
**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
DNA Flex - Clean-up Libraries
50m
50m
Clean-up Libraries Date/Initials:_________________
This step uses double-sided bead purification procedure to purify the amplified libraries.
50m
[ ] Centrifuge plate at 280 x g, Room temperature, 00:01:00 , and then place on magnet for 00:05:00 or until clear
[ ] Transfer 45 µL of supernatant to clean wells of a new midi plate (CED), and then remove from magnet
[ ] Vortex and invert SPB.
[ ] Add 81 µL of SPB** to samples, pipette 10
Note
**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.
times to mix.
[ ] Seal (CED) plate and incubate for at least 00:05:00 .
[ ] 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.
[ ] Place on magnet for 00:05:00 or until clear. Remove and discard supernatant.
[ ] Perform two 00:00:30 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.
[ ] Air dry beads approx. 00:05:00 and then remove plate from magnet
[ ] Vortex RSB and add 32 µL . Pipette to mix and incubate at Room temperature for 00:02:00 .
[ ] Place plate on magnet for 00:02:00 . Transfer 30 µL supernatant to new wells.
[ ] Library is now ready for quantification.
Note
SAFE STOPPING POINT
If 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 - Qubit Quantification
DNA Flex - Qubit Quantification
Qubit Quantification Date/Initials:_________________
[ ] Prepare Qubit working solution using Calculation 5. Label assay tubes for samples and standards.
| Reagent | Volume (uL) | *(#rxns+2std+2) | Lot# | Exp. Date | |
| Qubit Reagent | 1.0 | ||||
| Qubit Buffer | 199.0 | ||||
| Total | 200.0 |
Calculation 5: Qubit Working Solution
[ ] Combine 190 µL Qubit working solution + 10 µL Qubit standard into labeled standard tubes
[ ] Combine 198 µL Qubit working solution + 2 µL extracted DNA into labeled tubes
[ ] Vortex all tubes for 2-3 sec and incubate at Room temperature for minimum of 00:02:00 . Read tubes within 01:00:00
[ ] Record sample results.
DNA Flex - Denaturation of Pooled Library
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.
[ ] Centrifuge plate at 280 x g, Room temperature, 00:01:00
[ ] Make 4 nanomolar (nM) pool using Calculation 6
| 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) |
Calculation 6: 4nM Pool
[ ] Add 400 µL molecular grade H20 to the 100 µL 1.0 N NaOH aliquot for 0.2 N NaOH
[ ] Combine 5 µL 0.2 N NaOH and 5 µL pooled DNA in a 1.5 mL tube. Incubate 00:05:00 at Room temperature
[ ] Immediately add 990 µL pre-chilled HT1. Pipette to mix, then use Table 2 for desired library pool concentration
| Final Library Conc (pM) | 8 | 10 | 12 | 14 | 15 | 16 | 18 | 20 | |
| Pooled Library (uL) | 400.0 | 500.0 | 600.0 | 700.0 | 750.0 | 800.0 | 900.0 | 1000.0 | |
| HT1 (uL) | 600.0 | 500.0 | 400.0 | 300.0 | 250.0 | 200.0 | 100.0 | 0.0 |
Table 2: Pooled Library Dilution - Calculation for 4 nM pooled library/20 pM denatured pooled library