Coronavirus Disease 2019 (COVID-19) - Variant of Concern Screening and Whole Genome Sequencing Surveillance

Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.


  • As of February 6, 2023, SARS-COV-2 (COVID-19) Variant of Concern (VOC) PCR will no longer be performed at Public Health Ontario’s laboratory as this test no longer has any clinical, epidemiological or public health utility. Requests for COVID-19 VOC test will be rejected.
  • Specimens for representative surveillance will continue to be sequenced, see section below.

As of November 12, 2021 PHO’s laboratory is randomly selecting a proportion of all SARS-CoV-2 positive specimens, with Ct≤ 30* from the SARS-CoV-2 PCR diagnostic test, for whole genome sequencing (WGS).  This replaces the previous process implemented on May 26, 2021 for sequencing based on the VOC real-time PCR test.

The proportion of selected specimens will be adjusted periodically and determined according to positivity rates, the number of positive specimens, and testing capacity. When capacity allows, up to 100% of eligible specimens will be tested by WGS. This sampling method was first implemented on June 14, 2021, and aims to remove selection bias, provide better representation of known variants for surveillance, and allow improved detection of new emerging variants.

The following link provides the latest summary for SARS-CoV-2 Whole Genome Sequencing in Ontario

In addition to the selected proportion (if less than 100%), sequencing will be performed on specimens meeting the following criteria:

Routine sequencing:

  1. International travel – all specimens from travelers and contacts will be sequenced if travel is indicated on the COVID-19 diagnostic test requisition. If travel was not indicated on the requisition, testing can be added upon request.

For International travel, sequencing results will be sent as a standard report back to the ordering healthcare provider and to the public health unit for all specimens; however, only VOC lineages will be named on the report. Due to the dynamic nature of non-VOC lineage naming, lineages not currently designated as VOC will be reported back as “Variant of Concern not detected”. Similarly, Variants of Interest (VOIs) will not be identified as such on the laboratory report, nor will the lineage be reported. Descendent lineages of VOCs will be reported as the parent lineage only (e.g. AY.4 Delta descendent lineage reported as B.1.617.2).

As of December 23, 2021 all specimens indicating suspected vaccine breakthrough are no longer routinely sequenced for targeted surveillance. Vaccine breakthrough will continue to be monitored by sequencing through specimens identified as such in the proportional sampling described above.   

Sequencing by request:

  1. Suspected reinfection – sequencing can be requested to support the classification of a case as re-infection that does not meet criteria to be classified as a case of time-based reinfection as outlined in the Ontario COVID-19 Case Definition.
  2. Certain outbreak investigations – sequencing can be requested to support outbreak investigations.

For sequencing by request, the submitter may request a simple lineage-only report or in-depth genomic analysis.  If WGS testing is agreed to by the Ontario COVID-19 Genomic Network, submitters should confirm the specimen’s eligibility and availability with the original testing lab and arrange for specimens to be sent to the WGS testing laboratory. Please refer to the SARS-CoV-2 Whole Genome Sequencing Request guidance document and contact for support.

* The specimen’s SARS-CoV-2 PCR cycle threshold (Ct) value must be ≤ 30 for successful genome sequencing, although a subset of samples with a Ct ≤ 30 may not be successfully sequenced likely due to variability in determination of Ct values, RNA degradation, or sequencing technical issues.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

SARS-COV-2 Variant of Concern PCR Screening

SARS-CoV-2 PCR positive specimen with Ct value ≤ 30

1.5 ml preferred (min 1.0 ml)


Submission and Collection Notes


For travel or suspected reinfection whole genome sequencing requests, if the specimen is at PHO’s laboratory call Customer Service Centre (416-235-6556 or 1-877-604-4567) or fax the SARS–CoV-2 Variant of Concern Testing/Whole Genome Sequencing Requisition to (416-235-6552) to request to add the test; if the specimen is at another laboratory use the form above to send the specimen to PHO’s laboratory.


For outbreak investigations email to complete the request form and obtain approval/WGS-request ID before shipping any specimens. Submitters will receive information on signing up for a secure Incident and Outbreak Response Collaboration SharePoint Site account. This site has been authorized for the transfer of Personal Health Information and submitters will receive the in-depth genomic reports via this site.


This laboratory-developed investigational assay is being used for surveillance purposes and public health management. It is not a clinical diagnostic test.

Storage and Transport

Specimen should be maintained at 2-8oC during transportation (freeze if anticipated transport time to the laboratory is >72 hours).

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

SARS-CoV-2 genome sequencing results will be available within 10 days.

Test Methods

This laboratory-developed investigational assay is being used for surveillance purposes and public health management.

Sequencing involves PCR amplification, followed by sequencing of the majority of the approximately 30,000 base pair SARS-CoV-2 genome. Generated sequences are processed using bioinformatic tools and assigned a Pango lineage using the PANGOLIN software package, allowing for the identification of VOC and other lineages.


WGS Algorithm:

  1. A proportion of all SARS-CoV-2 positive specimens with Ct ≤ 30 will be selected for sequencing
  2. Specimens not already selected for sequencing that meet select conditions (e.g. international travel, outbreak) can be requested to be sequenced


WGS results will be reported to the ordering healthcare provider and the local Public Health Unit.

Data and Analysis

Updated 4 March 2024