SARS-CoV-2 Strain Sequencing
The UMGC has developed a PCR amplicon-based sequencing service that leverages protocols developed by the ARTIC Network, while reducing cost and increasing throughput. The details of our methodology have been published here. In brief, the method employs 95 PCR primers pairs to tile the SARS-CoV-2 genome with overlapping amplicons in a total of four multiplexed amplification reactions to achieve virtually complete genomic assembly.
We are offering this service to both UMN researchers and external researchers at cost recovery rates, in order to help accelerate the fight against the COVID-19 pandemic. The service is a “soup-to-nuts” workflow (including QC, library creation, sequencing, genomic assembly and annotation relative to reference, and (optional) upload to NextStrain.
This service takes extracted RNA from SARS-CoV-2 positive samples and uses a tiled amplicon approach to sequence the SARS-CoV-2 genome. An initial RT-qPCR quality control step using the protocol described here is performed to ensure that there is sufficient material for library preparation and sequencing.
We are currently recommending sequencing samples with N1 and N2 Ct values of 30 or less, which we estimate corresponds to ~100 SARS-CoV-2 genome copies per microliter. For samples that meet this criteria, we have observed average genome coverage values of 98.81% (at 10x) and 94.72% (at 100x) at a subsampled read depth of 100,000 raw reads.
|SARS-CoV-2 Tailed Amplicon UDI Sequencing1|
|Low Scale. Multiples of 12.2||12-36||sample||$80.676||$95.236|
|Standard Scale. Multiples of 48.3||48-3365||sample||$46.366||$54.666|
|High Scale. Multiples of 94.4||≥378||plate||$32.296||$37.376|
- The service includes a) quantification of SARS-CoV-2 using 3-reaction qRT-PCR as described in Nelson et al (2020) and b) QC report, followed by PCR-amplicon mediated sequencing as described in Gohl et al (2020), comprised of the following steps: c) reverse-transcription and library creation, d) sequencing via Illumina NGS, e) assembly and annotation of resulting reads, and f) (optional) upload of data to NextStrain. Raw sequencing results are also provided.
- Sequencing employs paired-end 2x300 bp reads on the Illumina MiSeq.
- Sequencing employs either paired-end 2x250 bp reads or 2x300-bp reads on the Illumina MiSeq.
- Sequencing employs paired-end 2x250 bp reads on the Illumina NovaSeq.
- Specifically, submissions are for 48, 94, 144, 188, 240, 282, or 336 samples.
- Price per sample assumes that all samples continue from submission through to sequencing. In the case that some samples fail QC and are removed from a sequencing batch, the batch price will remain the same, hence the per-sample price will be higher. Consequently, following QC, all clients will have the option of electing to a) pause a project in order to replace failing samples, or b) abandon the project at the QC step, for which the cost is $7.50 per sample. EXTERNAL rate is based on cost-recovery, whereas INTERNAL rate is reduced by UMN-specific subsidies.