SARS-COV2 SEQUENCING
ABOUT
With the advent of the covid-19 pandemic, the need arose for genomic surveillance to better understand the epidemiology, identify clusters and investigate outbreaks. To accomplish this, a partnership with IBC-ULB (Institut de Biologie Clinique ULB), ULB Erasme and the microbiology unit of UZ Brussel was initiated.
Since July 2021, BRIGHTcore has been sequencing Sars-Cov2 samples for diagnostic and research purposes.
METHODOLOGY
RNA is extracted from naso- and oropharyngeal swabs from Covid-positive patients (e.g. confirmed through qPCR). Samples, preferentially with a viral load > 3 log copies/ml are retained for sequencing.
The extracted RNA is converted to cDNA with a reverse transcriptase (NEB LunaScript) and subsequently used as input for a multiplex PCR (IDT Midnight PCR panel), to specifically enrich for the viral genome and reduce the human transcriptome background.
Next, the multiplex PCR amplicons are processed through the Oxford NanoPore Technologies Rapid Barcoding kit 96 (SQK-RBK110.96) and sequenced on the GridION x5 using a maximum of 96 samples per flow cell.
The full protocol from Nikki Freed et al. can be consulted here : full protocol. We slightly modified this protocol with additional (degenerate) primers as to compensate for PCR dropouts caused by the large degree of variations in the Omicron lineage.
More information on WGS and MPS is available through the buttons below.
INSTRUMENTS
The following devices are used for Sars-Cov2 sequencing in our premises.
NanoPore GridION x5
Sequencing long reads is no hassle on this device. It can sequence 5 MinION flowcells simultaneously and perform the data analysis on board.
CERTIFICATES
Sciensano recognized
DOWNLOADS
Sars-Cov2 SNV stats - PNG
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Verzendinstructies Sars-Cov2 RNA stalen - NL
2
Sars-Cov2 consensus fasta - fasta
1
Verzendinstructies Sars-Cov2 UTM stalen - NL
1
Sars-Cov2 coverage stats - PNG
1
Sars-Cov2 summary file - csv
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CONTACTS
BRIGHTcore general inquires
+32 (0)2 477 64 79 (sec)
UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
Toon Janssen
+32 (0)2 477 64 79 (sec)
UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
Ben Caljon
+32 (0)2 477 64 79 (sec)
UZ Brussel
BRIGHTcore
Laarbeeklaan 101
1090 Brussels
SAMPLE TYPES
We aim to keep the list with sample types updated. However, if you believe we offer this test on other sample types, or if you have a very specific sample type you'd like to evaluate : please contact the corresponding persons for either diagnostic or research purposes.
Tissue | Recipient | Quantity | Transport | Purpose / Extract |
|---|---|---|---|---|
Sars-Cov2 RNA (naso/oropharyngeal) | Microtube* or 96 wel plate (plate is preferred) | Log(copies/ml) > 3 (min. 10 µl) | Ice (max. 20 min) or dry ice | RNA |
Sars-Cov2 on UTM (naso/oropharyngeal) | UTM (Universal Transport Medium) or PBS tube w/o inactivation buffer | 500 µl (Ct<=25) | Room temperature | RNA |
* Microtube can be a either a cryovial or eppendorf tube (0,5 ; 1,5 or 2 ml). Please consider the quality of the tube used (nuclease free ; free of RNA/DNA ; sterile ; LoBind) according to application needed.
DELIVERABLES
Below, you can find the type of data files that can be retrieved for this test.
If you require more information or need a more custom output, please consult our Bioinformatics page.
Deliverable | Description | Research or diagnostic |
|---|---|---|
Sars-Cov2 consensus fasta | Consensus Sars-Cov2 genome (fasta format) for all samples in run/centre | Diagnostic / RUO |
Sars-Cov2 SNV stats | PNG file to visualize the quality of the individual SNVs on the report | Diagnostic / RUO |
Sars-Cov2 coverage stats | PNG file to visualize the completeness of Sars-Cov2 genome coverage | Diagnostic / RUO |
Sars-Cov2 summary file - csv | Comprehensive report with quality metrics and genotyping results | Diagnostic / RUO |
Raw data - fast5 file | Raw data file generated by Oxford NanoPore sequencers | RUO (on request) |
Annotated variant file | Text file containing all detected variants + relevant annotation (cross reference to various databases) | Diagnostic / RUO |
PLAN EXPERIMENT
1. For diagnostics
The decision to determine/sequence the full Sars-Cov2 genome is up to the microbiology or covid-lab, as this is only allowed for in specific cases/indications. As this is a diagnostic assay, the results are handled through your General Practitioner.
2. For Research
Alternatively, you may want to set up a study, e.g. to evaluate certain clusters, to assess immune evasion (efficacy of vaccines),... For this, please get clearance from your local ethical committee and/or register your samples with a biobank.
To set experiments up with us, please follow the steps below.
Get in touch (ONLY FOR NEW PROJECTS)
Explain your project, so we can assist you to find the best solution.
Expect questions like :
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What type of sample would you like to analyze?
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Will you handle the RNA extraction (please know : BRIGHTcore doesn't handle human pathogens)?
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In which elution buffer did you elute your RNA (we prefer nuclease free water)?
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How many samples would you like to process?
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How fast do you need the results (TAT) and/or do we need to expedite?
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If you deviate from our sample types, are there test samples available?
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Do you require bioinformatics support or are our standard deliverables ok?
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...
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Request quotation
Once you know which experiment you want to set up, fill out the 'Quote request' form, with the essential details of your project :
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Coordinates of the person to whom to address the quotation
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Which type of test you'd like to set up
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Do you require bioinformatics support?
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Do we need to expedite (comes at extra cost)?
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...
2
Submit your samples
Once you agree with our terms (quote / TAT / terms & conditions) :
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Fill out our sample submission form
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Send it via email : contact us
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Print out the form and include it with your samples
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Send your samples to us
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Adhere to the corresponding transport conditions listed under Sample types
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Label your tubes/plates correctly : see details here
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Please attach the 'verzendinstructies' (see downloads) on the shipping container (for RNA)
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Indicate if we can discard your samples after completion of project
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Your experiment starts
Now it is up to us... We will start your experiment as soon as possible. When expedited, we will start the experiment within 1 week. For standard requests the turn-around-time (TAT) varies :
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In diagnostics : 5 workdays
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In research : 4 weeks (or otherwise agreed)
The research TAT is generally overestimated, but this extended timeframe allows us to : combine similar requests of multiple scientists and to repeat the experiment in case of issues.
Please refrain from contacting us in case the pre-agreed TAT didn't expire yet.
