Welcome to Benchling!

You're looking at a Notebook entry. Here are some things you can do with it:

Track every step of your experiment

  1. Add new dates for every day your experiment runs.
  2. Capture protocols, observations, and tasks.
  3. Automatically keep a detailed history of your work.
  4. Auto-populate your calendar with Notebook entries.

Insert tables with calculations

Link sequences directly in your entry

Attach images and results

Share data with your collaborators

Create account

Sign up with GoogleI already have an account
Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. **It doesn't look like your current browser is supported** - for more information, [click here](/browsers).
Monospace normal
Monospace bold
Advanced Settings
  • 0 feature results
NGS003: Preliminary test of NGS Library Prep on successfully amplified cDNA from wastewater extracted RNA samples (open)
  • Notes
  • Metadata
  • Relevant Items
Editing disabled on read-only entry.

 
NGS003: Preliminary test of NGS Library Prep on successfully amplified cDNA from wastewater extracted RNA samples (open)

Tuesday, 9/7/2021AuthorsBackground and GoalChoosing what samples to do library prepAvailable ARTIC amplified samplesWednesday, 9/8/2021NGS003_R1 Shortlisted ARTIC cDNA samplesRun 1 executionDilution of adaptorsEnd polishing + A-tailingNEBNext End Prep instructions 1NEBNext End Prep instructions 2Adaptor ligationNGS003_R1 Adaptor ligation recipeNGS003_R1 Wells of E7395 kit used for barcoding cDNAThursday, 9/9/2021DNA cleanupPCR enrichment of adaptor ligated DNANEBNext PCR enrichment recipe for E7395NEBNext PCR thermocycling protocol for E7395NEB recommended cycle numbers of target enrichment library prepNGS003_R1 PCR number of cycles planningNGS003_R1 Planning PCR enrichment cycle #PCR product cleanupCheckingNGS003_R1.1_annotated_D1000_result.pngNGS003_R1.1 DNA amountNGS003_R1.1 library descriptionsFriday, 9/10/2021Monday, 9/13/2021Pooling the libraries for sequencingSample Requirement from Edinburgh GenomicsNGS003_R1 Final library mixingWednesday, 9/15/2021Tuesday, 9/21/202118713GNpool011_2021-09-16_14-40-20.pdfFriday, 9/24/2021Tuesday, 9/28/2021
Tuesday, 9/7/2021
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
Objective W8.1: Complete NGS libraries using cDNA amplified from wastewater samples
From ​Inaccessible Entry and ​Inaccessible Entry , we have successfully amplified some cDNA
from wastewater samples using the ARTIC and subARTIC v4.0 primers. With these cDNA, it would be a right time to attempt turning them into Illumina MiSeq compatible sequencing libraries.
Question to ask: ultimately the question is on what methods are best. But this experiment does not really answer that.
Rather, the issue is just to run through the process of sample prep and have an idea of what it's like.
Also importantly, to get some preliminary data so as to have an idea of the library complexity of the wastewater samples.
Choosing what samples to do library prep
Given this is the first time for me to perform this process, I intend to limit the number of libraries.
However, according to Microsoft Word - E7395 NEBNext Multiplex Oligos 96 Unique Dual Index UMI Adaptors DNA Set 1.docx, it is not recommended to go for < 4 samples.
It would be good to have > 8 samples so that regardless of how I pick the wells I could just use vertically down the plate.
Up to this point, the following samples are available:
*In cases where pre-purification amounts were reported, the D1000 tape result appear to strongly overestimate the total amount of DNA present inside the sample.
Wednesday, 9/8/2021
Thoughts on selecting samples
1.
Should get to know what EtOH precipitated samples were like, because they will inform if the process of EtOH was meaningful or not. In particular, samples of Exeter had very low concentrations and therefore precipitation almost appear to be a must.
2.
Samples from SEPA where we could amplify cDNA without EtOH precipitation or concentration are a must. These samples inform us what the actual data should look like.
3.
At least 1 EPC samples should be included for positive control
4.
Should aim for 8-10 samples
After thinking this over, decided to go for the following samples:
Run 1 execution
Dilution of adaptors
While the reaction is being done, need to dilute adaptors for wells C2-H2. These will be used for samples s05-s10.
Marion already took adaptors from wells A1-H1, A2-B2. Presumably she took 1.25 µL because she followed the adaptor ligation protocol of the NEB ARTIC kit.
This means she most likely took 1.25 μL.
Similarly, 1.2 μL will be taken for 10 fold dilution.
For 10 fold dilution
1.2 µL of adaptor will go in 10.8 μL of NEBNext UMI Adaptor Dilution Buffer.
End polishing + A-tailing
Follow the NEBnext End Prep protocol in the NEB kit for SARS-CoV-2 detection manual (E7650)
The total reaction volume is smaller
NEBNext End Prep instructions 1
Loading...
Loading...
NEBNext End Prep instructions 2
Loading...
Loading...
Adaptor ligation
This is the place when neither protocol from E7395 nor E7650 alone would work.
●
In the kits available, there was no ligation enhancer? we found the ligation enhancer
●
Adaptors with UMI are used
●
A smaller reaction volume is used following that of the ARTIC kit
●
It appears that, the E7650 protocol uses NEBNext adaptors for Illumina which are truncated and must be PCRed to add the full P5 and P7 sequences.
Those NEBNext adaptors also need the USER enzyme because the adaptors need to be cleaved prior to the PCR step.
Whereas, the E7395 protocol does not require this.
Mixing together these features the ligation recipe is rewritten as follow:
Note: the above master mix recipe in the end wasn't enough for all 10 samples. The last sample (s10) needed to be individually mixed again. The problem was that the mix was too viscous and during the pipetting process too much master mixes were lost to tips.
PCR program:
20 °C for 15 mins with heated lid off.
Then samples were stored for 1 overnight at -20 °C.
Thursday, 9/9/2021
DNA cleanup
Proceeded to the DNA clean up step.
NEB's protocol suggested doing size selection using NEBNext / SPRIselect beads.
After checking again, the NEB protocol probably assumes randomly fragmented DNA in the initial input.
But this is not our case, which we know most amplicons were 400 bp in sizes.
Therefore, it doesn't make sense to adopt the size selection protocol.
Rather, should move with the section 3B in Protocol for E7645, which is Cleanup of Adaptor-ligated DNA without size selection for all samples
The final volume from the last step is 46.75 µL. It will be necessary to top it up to 93.5 µL to continue following the NEB's protocol.
Added 46.75 µL of 0.1X TE for the top up.
Then followed the protocol onwards.
Carried out cleanup according to the protocol.
80% EtOH preparation:
For 10 samples, needed 0.4 mL * 10 = 4 mL minimum
Made 4.5 mL 80% EtOH by mixing 3.6 mL EtOH and 0.9 mL nuclease-free water.
PCR enrichment of adaptor ligated DNA
This part is basically identical to the protocol for E7395.
NEBNext PCR enrichment recipe for E7395
Loading...
Loading...
NEBNext PCR thermocycling protocol for E7395
Loading...
Loading...
The only problematic bit is the number of cycles that should be done.
In this experiment, I had multiple samples with vastly different input cDNA amount.
NEB has it's recommended thermocycling numbers for the PCR enrichment step.
NEB recommended cycle numbers of target enrichment library prep
Loading...
Loading...
Looking back at the samples, it would mean very different ranges of cycle numbers for different samples.
Therefore, the issue is how many PCR machines I could use at the same time and how I should group the samples.
This would require 4 PCR machines in total.
PCR product cleanup
Carried out cleanup according to the protocol.
80% EtOH preparation:
For 10 samples, needed 0.4 mL * 10 = 4 mL minimum
Made 4.5 mL 80% EtOH by mixing 3.6 mL EtOH and 0.9 mL nuclease-free water.
Checking
After final elution of 30 μL, took 0.5 μL from the library prep and diluted 1:10 into 4.5 μL of water.
Then QCed DNA using the TapeStation with D1000 tapes.
Most samples look good.
NGS003_R1.1_annotated_D1000_result.png
Loading...
Loading...
Some of the samples did not look clean. It is unsure if another cleanup is necessary (should be quite impossible judging from the SPRIselect guide) or whether we should just leave them out in the sequencing run.
Need input from Nick.
Friday, 9/10/2021
The decision is that the samples are real world samples and no further clean up should be done on them.
Monday, 9/13/2021
Pooling the libraries for sequencing
For the samples to be sequenced, they have to be pooled together in equimolar concentrations.
Aside from that, Edinburgh Genomics has specified what is the sample concentration and volume that needs to supplied.
*Use the following equation for nM = (ng per uL of DNA x 1,000,000) / (Number of base pairs x 660)
** Please contact us if you can't meet these requirements
So based on this, worked out how to mix the samples together.
Pooled the samples by adding the "Rounded volume" together. That would be the concentrated pool. It will probably be up to Edinburgh Genomics to work out how much they want to dilute the DNA prior to loading onto the flow cell. Stored pooled samples in -20 °C in the CWW wastewater sample box.
Wednesday, 9/15/2021
Put samples in tube arrived from Edinburgh Genomics and passed to Nahuel, who carried it to EG at King's Buildings.
Samples will be sequenced on a MiSeq sequencer with 250 PE.
Tuesday, 9/21/2021
Sample QC returned from Edinburgh Genomics.
18713GNpool011_2021-09-16_14-40-20.pdf
Proceeded with sequencing.
Friday, 9/24/2021
I decided to reallocate the sample library name and ID.
Tuesday, 9/28/2021
I decided that submission of the library concludes the experiment. Data analysis should probably has its own experiment.
NGS003 was completed and closed.

Welcome to Benchling!

Sign in to view data.

Split Workspace

Welcome to Benchling!

T
Tomasz has shared a file with you.