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NGS001: Attempting ARTIC SARS-CoV-2 cDNA amplification on concentrated wastewater (open)
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NGS001: Attempting ARTIC SARS-CoV-2 cDNA amplification on concentrated wastewater (open)

Wednesday, 8/25/2021AuthorsBackground and GoalRun 1 ExecutioncDNA synthesisNGS001_R1 cDNA samplesRecipe (copied from E7650 manual)Thermocycling protocol (copied from E7650 manual)cDNA amplificationcDNA amplification instructionscDNA amplification thermocyclingThursday, 8/26/2021NGS001_R1 TapeStation loadingNGS001_R1_annotated_HSD1000_result.pngFriday, 8/27/2021Monday, 8/30/2021Run 2 (NGS001_R2)NGS001_R2 samples (unused)NGS001_R2 samplesTuesday, 8/31/2021NGS001_R2 TapeStation loadingNGS001_R2_annotated_HSD1000_result.pngNGS001_R2 DNA amountRun 3 (NGS001_R3)NGS001_R3 samplesDilution schemeRTcDNA amplificationNGS001_R3 cDNA amplification recipeWednesday, 9/1/2021NGS001_R3_annotated_HSD1000_result.pngNGS001_R3 DNA amountThursday, 9/2/2021Run 4 (NGS001_R4)NGS001_R4 samplesFriday, 9/3/2021Table4NGS001_R4.pngNGS001_R4 TapeStation sample concentrationsRun 5 (NGS001_R5)Table3Monday, 9/6/2021NGS001_R5.pngTable5NGS001_R5_SEPA_259.pngNGS001_R5_WHIT_42.pngNGS001_R5_EPC.pngNGS001_R5_NTC.png
Wednesday, 8/25/2021
Authors
This is a copy of the original notebook created by Trevor Ho and Nahuel Moreno in Nick Gilbert group (https://chromatinlab.org/) for the COVID Wastewater Scotland project (https://biordm.github.io/COVID-Wastewater-Scotland/)
Background and Goal
Object W6.1: Working on SARS-CoV-2 library prep using NEB ARTIC primers.
We will start the NGS part of this work by trying to amplify SARS-CoV-2 cDNA from wastewater using the complete set of ARTIC primers V3 (produced by NEB).
There are 2 kits from NEB:
The steps are essentially the same except library prep.
For this experiment we only care about whether it is possible to amplify cDNA from the library. So there is no need to look into the difference for the downstream processes.
The instructions for carrying out the reactions are detailed here:
There are the Express Prep and Standard Prep protocol. Stuck to the standard prep protocol because Express Prep protocol expects viral genome copy number to be > 100. We made no such assumptions.
Note that all purified, ARTIC amplified cDNA were 1:10 fold diluted before being loaded onto the TapeStation, as stipulated in NEB's manual.
Run 1 Execution
cDNA synthesis
4 samples will be used:
Recipe (copied from E7650 manual)
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Mixed reactions for reverse transcription individually.
Thermocycling protocol (copied from E7650 manual)
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cDNA amplification
cDNA amplification instructions
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Simply followed instructions as above. All tubes were added separately.
cDNA amplification thermocycling
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After the reaction is done. Did not proceed to purification by magnetic beads straight away. Instead, moved onto TapeStation measurement.
Change of plan, decided to proceed with bead purification.
Pooled amplified cDNA from pool 1 and pool 2 together (pipetting everything I could get from pool 2 reactions into pool 1)
Then stored the amplified cDNA in -20 °C.
Thursday, 8/26/2021
Proceeded to bead purification. Followed the protocol till step 2.3.12.
To make 80% EtOH for purification, made 2 mL by adding 0.4 mL nuclease free water to 1.6 mL EtOH.
Used the High sensitivity D1000 tape and measured 1/10 diluted pooled cDNA samples:
Result:
NGS001_R1_annotated_HSD1000_result.png
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Conclusion: the NEB ARTIC kit doesn't work with wastewater samples.
Possible issues:
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Samples are too dirty even after EtOH precipitation
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Samples are too degraded and there was just no products with sufficiently large sizes to cover the region of interest
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Concentrated samples are too concentrated (is this even sensible though?)
So many irrelevant RNA molecules get reverse transcribed and used up a lot of resources during the unspecific RT-step?
They outcompeted the heavily degraded SARS-CoV-2 RNA in the RT-step??
Friday, 8/27/2021
Ways to test the hypotheses
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Northern blot of RNA samples (need to keep asking Jon)
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Spike unconcentrated and concentrated samples with EPC and try doing the RT-PCR again
Monday, 8/30/2021
Run 2 (NGS001_R2)
Objective W7.1: Spike wastewater samples with EPC. Then try doing cDNA amplification from spiked samples.
First, sample volumes for each RT reaction was 8 µL. Therefore, need to decide how to spike the samples.
The balance is to ensure enough EPC goes into the sample while the samples is still dominantly representing it.
Therefore, decided to try spiking 2 µL of stock EPC into 6 µL of RNA sample, whether diluted or undiluted.
This ratio was chosen arbitrarily.
With that done, decided to follow same protocol as Run 1.
For this run, used fresh samples for both unconcentrated Exeter and SEPA samples.
Decided not to use concentrated SEPA samples because the amount remaining was just so limited. (20 µL eluted RNA and used 8 µL in NGS001_R1)
Also, primers for subARTIC just arrived and didn't want to risk using them up.
Changed this to:
It's a shame I (​Trevor Y. H. Ho ) should have noted down the labels of the Exeter and SEPA samples used.
Carried out the steps up to cDNA amplification
Tuesday, 8/31/2021
Proceeded to bead purification
Needed 0.4 mL of 80% EtOH for each sample, so need 8 * 0.4 mL = 3.2 mL of 80% EtOH.
Added 0.7 mL of nuclease-free water to 2.8 mL EtOH.
NGS001_R2_annotated_HSD1000_result.png
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Then why can't we amplify cDNA from concentrated samples using ARTIC primers?
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Concentrated too much in the process leading to too many junk RNA being RT-ed
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In the process we also concentrated PCR inhibitors
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RNA were damaged in the process of EtOH purification
Run 3 (NGS001_R3)
Situation:
Concentrated samples: no cDNA amplification observed
Diluted samples: weak cDNA amplification observed
Meaning: there should be a concentration between the fully concentrated samples and the diluted samples where cDNA amplification will give stronger result
Or RNA were simply damaged during concentration
Therefore, main goal is to serially dilute concentrated wastewater RNA samples and then look at ARTIC amplification.
Could possibly do a 1:5 or 1:10 dilution gradient
Also, would be good to assay if result of EtOH precipitation inhibit RT and cDNA amplification, so should also do EPC spiking at highest concentration tested here.
Result from NGS001_R2 suggested that I could possibly decrease the amount of EPC spiked in.
Decided to use 1 μL of EPC for spiking and 7 μL of samples
This would allow entire tape to be used.
Dilution scheme
Remaining sample volume should be < 12 µL.
To ensure minimum of 8 µL for each dilution, need to have 20 µL at each stage. So that 4 µL can go to the next 16 µL of water.
Best push it to 24 µL for each stage then.
Then each dilution would be 6 µL into 18 µL of water.
Remaining volume would be 18 µL, sufficient for 2 rounds of cDNA amplification in case it was successful.
So for 1st dilution: Add 16 µL of nuclease-free water to remaining concentrated sample RNA from Run 1.
This would give ~ 28 µL of sample.
Take 6 µL to dilute into 18 µL of water.
Repeat for 5 times.
After this is done, there should be 22 µL of most concentrated samples left, enough for spiking and direct loading.
RNA amount calculation.
For each sample of X ng/µL. It would be X * 12 / (12 + 16) for stock.
RT
Set up RT reaction by adding LunaScript SuperMix individually
cDNA amplification
In Run 1 and Run 2, cDNA amplification were set up for each tube individually.
This was too slow. Need to switch over to making master mix.
Set up cDNA amplification and ran the reaction overnight.
Wednesday, 9/1/2021
Proceeded to bead purification
Needed 0.4 mL of 80% EtOH for each sample, so need 16 * 0.4 mL = 6.4 mL of 80% EtOH.
Added 1.3 mL of nuclease-free water to 5.2 mL EtOH.
Completed the purification and loaded samples onto TapeStation.
NGS001_R3_annotated_HSD1000_result.png
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Observations and conclusions:
1.
EtOH precipitation must have ended up with RT or PCR inhibitors, because spiked most concentrated, samples did not work. Whereas in NGS001_R2, spiked RNA samples worked well
2.
Most concentrated samples also did not gave amplification products but increasing dilution of samples gave amplification products. For both Exeter and SEPA samples, the samples that yielded most cDNA products were at around 40 - 50 ng of input RNA amount.
3.
Comparing Run2 and Run3 of this experiment, in terms of PCR product amount quantified from TapeStation:
Run 2: Lane H1, SEPA samples unconcentrated: 212 pg/µL for the 400 bp band
Run 3: Lane E2, EtOH precipitated SEPA samples diluted by 25 fold: 222 pg/µL for the 400 bp band.
Therefore, for SEPA samples there was no a real benefit in concentrating the samples
4.
There is a benefit for Exeter samples though, because clearly a stronger band showed up from EtOH concentrated samples compared to unconcentrated sample (Run 2, Lane F1).
Therefore, for the next test to try subARTIC primers, it would be best to try the concentrated samples that were known to work with ARTIC primers and the NEB kit.
So far this experiment could be considered done but would be best to get Nick's comment on whether to proceed with library prep with samples that appeared to have worked.
This experiment also used a lot of Tapes at the recommendation of NEB. Would be good to switch over to agarose gel for subsequent analysis, but probably would need to load entire sample into the gel.
Also, the concentration measured from TapeStation is very close to the sensitivity limit of D1000 tapes (0.1 ng/µL) 5991-2706EN.indd (agilent.com), it would be also hard to use the normal D1000 tapes for this purpose.
Thursday, 9/2/2021
Run 4 (NGS001_R4)
Intended to replicate the results from run 2 on waste water samples, where we observed some weak but present bands of ~400bp. Used six samples: 2 SEPA, 2 Birmingham and 2 Exeter plus EPC and NTC.
Brought all fresh samples for Birmingham and Exeter, reused pre-existing SEPA samples from -20 freezer. SEPA sample 96 is a pool of other samples. SEPA sample 259 is presumed a single sample but it is not certain.
Set up RT-PCR as per kit specifications and followed up with cDNA amplification step. This will be left overnight and the protocol will be resumed the following day.
Friday, 9/3/2021
NGS001_R4.png
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I (​Nahuel) have potentially missplaced the EPC in one of the steps of the protocol, as there is no signal from well H1 and there is a big signal from well C1, which is in a mirrored position to H1 in the tube strip. I will have to repeat the library generation process.
There are some bands in all the waste water samples, which is promising. The library will be repeated and results will be compared with those.
Run 5 (NGS001_R5)
This run intends to correct for the sample missplacement in run 5.
It is run as a replicate of run 4 with the available samples from that run.
Samples:
Not enough SEPA sample left, it will be topped out with water to a total volume of 8 µL. Approx. 2 µL of water in 6 µL of sample.
Birmingham BHAM 7.2 4 was used up for NGS002 so it cannot be repeated.
Reactions were set to run in the evening and will be collected on Monday.
Monday, 9/6/2021
The samples were run on the TapeStation for DNA quantification.
NGS001_R5.png
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This time it shows that SEPA 259 seems to have a very high viral load. Therefore two options could have been possible last run: either EPC was lost during some of the steps, or EPC and SEPA 259 were combined accidentally in one of the steps.
The concentrations are lower for all of the samples. This could be due to leaving the beads to air dry for too long. There was some leftover liquid in the tubes which had to be pippetted out before adding the water for elution, which might have meant that samples were left for too long in this step.
In R4, all samples display bands at 400 bp suggesting that the amplification of the cDNA with the ARTIC primers was succesful. This replicate only SEPA 259 and Exeter WHIT 4/2 display consistent bands. There is a very faint band visible in the Birmingham sample as well.
NGS001_R5_SEPA_259.png
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NGS001_R5_WHIT_42.png
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NGS001_R5_EPC.png
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A band is seen in NTC at 200bp. This cannot be a product of the ARTIC primers but can be an indicator of potential contamination. This band appears very faintly (but undetected in the assay) in other samples excluding BHAM 7.2 8.
NGS001_R5_NTC.png
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After chatting with David from SEPA, sample 259 does not contain a particularly high viral load. We do not have an answer as why we see such a high amplification from it. We will proceed with the library generation and analyse the obtained results.
Run 5 confirms that the results from runs 4 and 5 are consistent with those obtained from run 2. We are able to amplify cDNA from waste water samples using ARTIC primers.
This experiment is now closed.

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