Mar 07, 2022
Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples
- Padmini Ramachandran1,
- Tamara Walsky1,
- Amanda Windsor1,
- Maria Hoffmann1,
- Chris Grim1
- 1FDA CFSAN
Protocol Citation: Padmini Ramachandran, Tamara Walsky, Amanda Windsor, Maria Hoffmann, Chris Grim 2022. Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples. protocols.io https://dx.doi.org/10.17504/protocols.io.b34qqqvw
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
Created: January 20, 2022
Last Modified: March 07, 2022
Protocol Integer ID: 57200
Keywords: SARS-CoV-2, Library Prep, NEB, COVID, VarSkip Short, wastewater, optimal variant detection from wastewater, wastewater samples purpose, wastewater sample, wastewater, wastewater collection, rna extraction, containing sar, genome sequencing, cdna synthesis, varskip short sar, applied nutrition for genometrakr, protocol details methods for cdna synthesis, data submission to ncbi, genometrakr, rna, monitoring sar, examining cdna amplicon sample, ncbi, sequencing, pandemic response project, sequenced fragment
Disclaimer
This method is under development and assessment for suitability of use. It is likely that modifications will be made to improve the method.
Abstract
PURPOSE:
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI.
This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2. The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications. Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers. These primers are available in the NEBNext®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep.
There are a couple of decision points in this protocol. Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions.
Guidelines
Overview
The NEBNext SARS-CoV-2 Library Prep Kit (Illumina) contains the enzymes, buffers and oligos required to convert a broad range of total RNA input amounts into targeted, high quality libraries for next-generation sequencing on the Illumina platform. Primers targeting the human EDF1 (NEBNext ARTIC Human Primer Mix 1) and NEDD8 (NEBNext ARTIC Human Primer Mix 2) genes are supplied as optional internal controls. The fast, user-friendly workflow also has minimal hands-on time.
Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of an indexed library on the Illumina sequencing platform.
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact [email protected] for further information.
Figure 1. Workflow demonstrating the use of NEBNext SARS-CoV-2 Library Prep Kit for Illumina
Materials
Kit:
NEBNext®ARTIC SARS-CoV-2 RT-PCR Module- E7626S/L-
This kit includes two sets of for primers:
- The VarSkip (for Variant Skip) Short primers have been designed at NEB to provide improved performance with SARS-CoV-2 variants, including the Omicron and Delta variants. The Omicron variant can be called confidently using NEBNext VarSkip Short (VSS) primers. Note that there are two dropouts (amplicons 56 and 67), and two amplicons (20 and 64) have lower coverage. Beginning 2/14/2022, NEB has released v.2 of VSS primers, which are also compatible, and preferred, with this protocol.
- The V3 ARTIC primers have been balanced, using methodology developed at NEB based on empirical data from sequencing. In combination with optimized reagents for RT-PCR, the kits deliver improved uniformity of amplicon yields from gRNA across a wide copy number range. (not used)
Once the amplicon is generated, the library prep of the amplicons can be done with either/or
Option 1) NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®)- E7650S/L
Option 2) Illumina® DNA Prep, (M) Tagmentation (24/96 Samples) 20018704/20018705
The Library Kit Includes:
The volumes provided are sufficient for preparation of up to 24 reactions (NEB #E7650S) and 96 reactions (NEB #E7650L).
Package 1: Store at -20 °C
- LunaScript™ RT SuperMix (E7651) — Store at -20°C Q5®
- Hot Start High-Fidelity 2X Master Mix (E7652) — Store at -20°C
- NEBNext® ARTIC SARS-CoV-2 Primer Mix 1 (E7725) — Store at -20°C (not used)
- NEBNext® ARTIC SARS-CoV-2 Primer Mix 2 (E7726) — Store at -20°C (not used)
- NEBNext® ARTIC Human Control Primer Pairs 1 (E7727) — Store at -20°C (not used)
- NEBNext® ARTIC Human Control Primer Pairs 2 (E7728) — Store at -20°C (not used)
- NEBNext® Ultra II™ End Prep Enzyme Mix (E7653) — Store at -20°C
- NEBNext® Ultra II™ End Prep Reaction Buffer (E7654) — Store at -20°C
- NEBNext® Ultra II™ Ligation Master Mix (E7655) — Store at -20°C
- NEBNext® Library PCR Master Mix (E7656) — Store at -20°C
- 0.1X TE (E7657) — Store at -20°C
- Nuclease-free Water (E7667) — Store at -20°C
Package 2: Store at Room temperature . Do not freeze.
NEBNext Sample Purification Beads
Required Materials Not Included:
- 80% Ethanol (freshly prepared)
- Nuclease-free Water
- DNA LoBind Tubes (Eppendorf #022431021)
- Magnetic rack/stand (NEB #S1515, Alpaqua®, cat. #A001322 or equivalent)
- Thermocycler
- Vortex Mixer
- Microcentrifuge
- Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables
- DNase RNase free PCR strip tubes (USA Scientific 1402-1708)
Either one of the index kit for Illumina DNA prep
- IDT® for Illumina® DNA/RNA UD Indexes Set A, Tagmentation (96 Indexes, 96 Samples) 20027213
- IDT® for Illumina® DNA/RNA UD Indexes Set B, Tagmentation (96 Indexes, 96 Samples) 20027214
- IDT® for Illumina Nextera DNA Unique Dual Indexes Set C (96 Indexes, 96 Samples) 20027215
- IDT® for Illumina Nextera DNA Unique Dual Indexes Set D (96 Indexes, 96 Samples) 20027216
Troubleshooting
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Before start
Note: The amount of RNA required for detection depends on the abundance of the RNA of interest. In general, we recommend, using ≥ 10 copies of the (SARS-CoV-2) viral genome as input, however, results may vary depending on the quality of the input. In addition, we recommend setting up a no template control reaction and that reactions are set-up in a hood.
The presence of carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.
cDNA Synthesis
Note
The presence of genomic DNA or carry-over products can interfere with sequencing accuracy, particularly for low copy targets. Therefore, it is important to carry out the appropriate no template control (NTC) reactions to demonstrate that positive reactions are meaningful.
We have also verified cDNA synthesis using the Invitrogen™ SuperScript™ IV First-Strand Synthesis System (Catalog number:18091200), as described in the SNAP protocol with modifications (random hexamers, RT incubation of 30 min.).
Gently mix and spin down the LunaScript RT SuperMix reagent. Prepare the cDNA synthesis reaction as described below:
| A | B | |
| COMPONENT | VOLUME | |
| RNA Sample | 8 µl | |
| (lilac) LunaScript RT SuperMix | 2 µl | |
| Total Volume | 10 µl |
For no template controls, mix the following components:
| A | B | |
| COMPONENT | VOLUME | |
| (white) Nuclease-free Water | 8 µl | |
| (lilac) LunaScript RT SuperMix | 2 µl | |
| Total Volume | 10 µl |
Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
Incubate reactions in a thermocycler* with the following steps:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Primer Annealing | 25°C | 2 minutes | 1 | |
| cDNA Synthesis | 55°C | 20 minutes | ||
| Heat Inactivation | 95°C | 1 minute | ||
| Hold | 4°C | ∞ |
*Set heated lid to 105°C
Note
Samples can be stored at -20 °C for up to a week.
Targeted cDNA Amplification
Note
4.5 µl cDNA input is recommended. If using less than 4.5 µl of cDNA, add nuclease-free water to a final volume of 4.5 µl. We recommend setting up the cDNA synthesis and cDNA amplification reactions in different rooms to minimize cross-contamination of future reactions. The cDNA reaction can be set up in hood to minimize cross-contamination.
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:
For Pool Set A:
| A | B | |
| COMPONENT | VOLUME | |
| cDNA (Step 2) | 4.5 µl | |
| (lilac) Q5 Hot Start High-Fidelity 2X Master Mix | 6.25 µl | |
| NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 | 1.75 µl | |
| Total Volume | 12.5 µl |
For Pool Set B:
| A | B | |
| COMPONENT | VOLUME | |
| cDNA (Step 2) | 4.5 µl | |
| (lilac) Q5 Hot Start High-Fidelity 2X Master Mix | 6.25 µl | |
| NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2 | 1.75 µl | |
| Total Volume | 12.5 µl |
Flick the tubes or pipet up and down 10 times to mix followed by a quick spin.
Incubate reactions in a thermocycler* with the following steps:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 30 seconds | 1 | |
| Denature | 95°C | 15 seconds | 35 | |
| Annealing/Extension | 63°C | 5 minutes | ||
| Hold | 4°C | ∞ | 1 |
*Set heated lid to 105°C
Combine the Pool A and Pool B PCR reactions for each sample.
Note
Samples can be stored at -20 °C for up to a week.
Assess the quantity of the cDNA amplicon using the Qubit HS kit. (1 µL each combined pool
Note
Based on the viral load in the sample, you will see a spectrum of fragment profiles. If there is a single peak around 400-600 bp, the clean-up step can be omitted and you can proceed directly to library preparation using Illumina DNA Prep
If there is not a single peak, check the amplicon size distributions. If there are lots of smaller peaks, for example, we typically see one at 44, 75, 128, and 213, these PCR reaction pools need to be cleaned up. Follow the cleanup protocol and prepare libraries using the Ultra II DNA workflow by NEB Next ARTIC SARS-CoV-2 Library Prep Kit (Illumina). If you are not able to perform the PCR pool assessment above, proceed to PCR cleanup and prepare libraries using the Ultra II DNA workflow by NEB Next ARTIC SARS-CoV-2 Library Prep Kit (Illumina).
Cleanup of cDNA Amplicons.
Note
The volume of NEBNext Sample Purification Beads provided here are for use with the sample composition at this step (25 µl; Step 6). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.
Vortex the NEBNext Sample Purification Beads to resuspend.
Add 20 µL (0.8X) resuspended beads to the combined 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 at Room temperature for at least 00:05:00 .
5m
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard the beads.
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
Perform second Ethanol wash:
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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.
30s
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 28 µL 0.1X TE . If not assessing cDNA (Step 24) elute DNA in 27 µL 0.1X TE .
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes 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 26 µL to a new PCR tube. If not assessing cDNA (Step 24) transfer 25 µL to a new PCR tube.
We recommend assessing cDNA amplicon (from Step 23) concentrations with a Qubit fluorometer using Qubit HS DNA assay kit.
Note
Amplicons may also be run on a Bioanalyzer or TapeStation® to confirm ~500 bp size of amplicons. To run on a Bioanalyzer, dilute amplicon 10-fold with 0.1X TE Buffer and run 2 µL on a DNA High Sensitivity ScreenTape. (See Figure 19 below for example of amplicon size profile on a Tapestation).
Figure 19: Example of cDNA amplicons generated from 1000 genome copies of SARS CoV-2 in the absence (A) and presence (B) of human primer controls.
Note
Samples can be stored at -20 °C for up to a week.
Quality checks on cleaned amplicons before proceeding to library prep
We have observed the following output for VSS amplicons based on viral titer in the wastewater samples:
1)Moderate to high viral titer samples: The cleaned amplicon quantity is expected to be 0.5 ng/µl to 8 ng/µl
2)Low viral titer samples: The cleaned amplicon quantity is expected to be 0 - 0.2 ng/µl
We recommend to proceed to library preparation for samples at 0.2 ng/µl or higher. Cleaned VSS amplicons below this value have not yielded sequencing results.
We recommend assessing cDNA amplicon (from Step 23) on Tape Station or Bioanalyzer using High Sensitivity kit.
Note
Figure 20: Tape station profile of cleaned amplicon from a moderate to high positive sample.
Figure 21: Tape station profile of cleaned amplicon from a low positive sample.
Figure 22: Tape station profile of cleaned amplicon from a low positive sample measured at ~0 ng/ul.
NEBNext End Prep
This is library preparation Option 1, and the most common that will be used. You will use the Ultra II DNA workflow as described in the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) (cat# E7650S/L), with NEBNext®Multiplex Oligos for Illumina®(96 Unique Dual Index Primer Pairs) (cat# E6440S/L).
Add the following components to a sterile nuclease-free tube:
| A | B | |
| COMPONENT | VOLUME | |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 1.5 µl | |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 3.5 µl | |
| Targeted cDNA Amplicons* | 25 µl | |
| Total Volume | 30 µl |
*When cleanup of the pooled cDNA amplicons is skipped, the amplicons must be diluted prior to library prep, please dilute the cDNA as mentioned here. Transfer 2.5 µl of the pooled cDNA amplicons to a fresh tube. Add 22.5 µl of 0.1X TE for a final volume of 25 µl. If cleanup is performed, use the cleaned targeted cDNA amplicons as the input.
Set a 100 µl or 200 µl pipette to 25 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.
Note
It is important to mix well. The presence of a small amount of bubbles will not interfere with performance
Place in a thermocycler* and run the following program:
| A | B | |
| TEMP | TIME | |
| 20°C | 30 minutes | |
| 65°C | 30 minutes | |
| 4°C | ∞ |
*Set heated lid to 75°C
Note
If necessary, samples can be stored at -20 °C ; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
Adaptor Ligation
Add the following components directly to the End Prep Reaction Mixture:
| A | B | |
| COMPONENT | VOLUME | |
| End Prep Reaction Mixture (previous step) | 30 µl | |
| (red) NEBNext Adaptor for Illumina** | 1.25 µl | |
| (red) NEBNext Ultra II Ligation Master Mix* | 15 µl | |
| Total Volume | 46.25 µl |
* Mix the Ultra II Ligation Master Mix by pipetting up and down several times prior to adding to the reaction.
** The NEBNext adaptor is provided in NEBNext Oligo kits. NEB has several oligo options which are supplied separately from the library prep kit. Please see www.neb.com/oligos for additional information.
Note
Do not premix adaptor with the Ligation Master Mix.
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.
Note
Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.
Incubate at 20 °C for 00:15:00 in a thermocycler with the heated lid off.
15m
Add 1.5 µL (red or blue) USER® Enzyme to the ligation mixture from the previous step.
Note
Steps 26 and 27 are only required for use with NEBNext non-indexed Adaptors. USER enzyme can be found in the NEBNext Multiplex Oligos (www.neb.com/oligos).
Mix well and incubate at 37 °C for 00:15:00 with the heated lid set to ≥ 47 °C .
Note
Samples can be stored overnight at -20 °C .
15m
Cleanup of Adaptor-ligated DNA
The following section is for cleanup of the ligation reaction.
Note
The volume of NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step (47.75 µl; Step 27). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.
Vortex the NEBNext Sample Purification Beads to resuspend.
Add 43 µL (0.9X) resuspended beads to the Adaptor Ligation 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 00:05:00 at Room temperature .
5m
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard beads.
Add 200 µL 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
Repeat the previous step once for a total of two washes:
Add 200 µL 80% freshly prepared ethanol to the tube/ plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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.
30s
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 target. 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 10 µL 0.1X TE .
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 00:02:00 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.
2m
Place the tube/plate on the magnetic stand. After 00:05:00 (or when the solution is clear), transfer 7.5 µL to a new PCR tube.
Note
Samples can be stored at -20 °C .
5m
PCR Enrichment of Adaptor-ligated DNA
Note
We only recommend using NEBNext Oligo kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied ay 10 µM (combined). We observed addtion of spurious nucleotides when using primers supplied in separate tubes
Add the following components to a sterile strip tube:
| A | B | |
| COMPONENT | VOLUME | |
| Adaptor Ligated DNA Fragments (Step 45) | 7.5 μl | |
| (blue) NEBNext Library PCR Master Mix | 12.5 μl | |
| Index Primer Mix (E6440S)* | 5.0 μl | |
| Total Volume | 25 μl |
* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.
Set a 100 μl pipette to 20 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 on a thermocycler** and perform PCR amplification using the following PCR cycling conditions:
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 98°C | 30 seconds | 1 | |
| Denaturation | 98°C | 10 seconds | 6-8* | |
| Annealing/Extension | 65°C | 75 seconds | ||
| Final Extension | 65°C | 5 minutes | 1 | |
| Hold | 4°C | ∞ |
* The number of PCR cycles recommended should be viewed as a starting point and may need to be optimized for particular sample types. If you have cleaned up cDNA as input, use 8 cycles. If you have diluted cDNA as input use 6/7 cycles.
**Set heated lid to 105°C.
Cleanup of PCR Reaction
8m
Note
The NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions the volumes may need to be experimentally determined.
Vortex NEBNext Sample Purification Beads to resuspend.
Add 22.5 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 00:05:00 at Room temperature .
5m
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Note
Caution: do not discard the beads.
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
30s
Repeat the previous step once for a total of two washes:
Add 200 µL 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at Room temperature for 00:00:30 , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
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.
30s
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 17 µL 0.1X TE .
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 00:02:00 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.
2m
Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 µL to a new PCR tube and store at -20 °C .
Check the size distribution on an Agilent Bioanalyzer or TapeStation. The sample may need to be diluted before loading. A peak size of ~520 bp is expected (Figure 56).
Note
Samples can be stored at -20 °C or proceed to MiSeq denature and dilution guideline to load it on to the MiSeq.
Figure 56: Example of final library size distributions on a TapeStation. ARTIC SARS-CoV-2 libraries
were generated from 1000 viral copies in the absence (A) or presence (B) of the human primer controls.
Sample Sheet generation for NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) with E6440 Index plate.
E6440_ Forward Strand Workflow Sample Sheet.csv
E6440_ Forward Strand Workflow Sample Sheet.csv