QIAseq SARS-CoV-2 Primer Panel

• 高感度で正確なSARS-CoV-2 RNA の逆転写およびターゲット増幅
• QIAseq FX DNA Library Unique Dual Index（UDI）Kit との併用で最大384サンプルまでマルチプレックスが可能
• わずか3～3.5時間のワークフローを最小限の作業工程で実現

QIAseq SARS-CoV-2 Primer Panel は、新型コロナウィルス感染症（COVID-19）を引き起こすSARS-CoV-2 の疫学的研究のために特別にデザインされたキットです。本キットをQIAseq FX DNA Library UDI Kit と併用することで、新型コロナウィルスSARS-CoV-2 ゲノム全体のPCR によるターゲット増幅およびNGS 用ライブラリー構築を可能にします。SARS-CoV-2 ゲノムはポジティブセンスの一本鎖RNA です。本キットには、SARS-CoV-2 ゲノムを特異的に増幅するPCR プライマーが含まれているため、SARS-CoV-2 RNA と宿主RNAが混合したトータルRNA サンプルからcDNA に逆転写した後、SARS-CoV-2 ゲノムを特異的に増幅することが可能です。本キットは、29.9 kb のSARS-CoV-2 ゲノム全体をカバーする200 を超えるプライマーペアで構成されています。

High-quality, single-box solution

Targeting SARS-CoV-2 requires both reverse transcription and whole genome enrichment of the viral RNA. The QIAseq SARS-CoV-2 Primer Panel combines both of these steps to generate amplicons for downstream library creation. When paired with a QIAseq FX DNA Library UDI Kit, you can construct sequencing-ready libraries compatible with Illumina platforms.

Community primer design

QIAseq SARS-CoV-2 primer pools utilize designs from the ARTIC V3 primers. The primers have also gone through an in silico check to reduce chances for dimerization during sample enrichment.

Multiplexing

Combine with a QIAseq FX DNA Library UDI Kit to enable multiplexing on high-throughput Illumina instruments, such as the NovaSeq (up to 384 samples per flow cell).

Data analysis

When paired with QIAGEN CLC Genomics Workbench, the panel can deliver data that can be quickly analyzed. Sequence data can be used to identify variants across different samples, as well as compare it to multiple genomes – from consensus reference genomes to one of many genomes that have been uploaded from around the world.

Viruses consist of nucleic acid (viral genome) and a limited number of proteins that facilitate entry into the host cell, replication of the genome and production of virions. While viral genomes can be comprised of RNA or DNA, SARS-CoV-2 is encoded by an RNA molecule. The size of the entire SARS-CoV-2 genome is under 30 kb and can be mixed with host RNA when isolating from a human sample, making it challenging to reconstruct the whole genome of the virus.

While next-generation sequencing (NGS) has become a vital tool, library preparation remains a key bottleneck in the NGS workflow. The QIAseq SARS-CoV-2 Primer Panel is a multiplex PCR primer set for whole genome amplification of SARS-CoV-2. Using primer pools that utilize designs from ARTIC V3 primers, the QIAseq SARS-CoV-2 Primer Panel generates amplicons that cover the entire SARS-CoV-2 genome. Combine with a QIAseq FX DNA Library UDI Kit to convert the amplicons into NGS-ready fragments compatible with lllumina sequencers.

From cDNA synthesis to enrichment

The QIAseq SARS-CoV-2 Primer Panel workflow begins with converting total viral RNA into cDNA using random priming (no rRNA depletion or poly-A selection is required). This reaction is flexible with regards to input RNA used for the reverse transcription reaction. To elaborate, 5 µl viral RNA input is required as a starting point. When the viral RNA has been previously assessed using a qPCR assay, the C_T value should be between 18–35. If the C_T is between 12–15, then dilute the sample 100-fold in water; if it's between 15–18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.

Following cDNA synthesis, primer pools (based on sequences from the ARTIC Network) are used in a high-fidelity multiplex PCR to prepare two pools of 400 bp QIAseq SARS-CoV-2 Primer Panel amplicons. The two enriched pools per sample are then combined into a single tube and then purified. This is then followed by QIAseq FX DNA library construction.

QIAseq FX DNA Library construction

Purified amplicons from a multiplex QIAseq SARS-CoV-2 Primer Panel are converted to Illumina-compatible NGS libraries using our QIAseq FX DNA Library UDI Kits, which provide a fast, fully enzymatic procedure from DNA fragmentation to NGS library.

Purified, target-enriched samples from the QIAseq SARS-CoV-2 Primer Panel are first enzymatically sheared into smaller fragments. The desired median fragment size is 250 bp. The fragmented DNA is then directly end-repaired and an "A" is added to the 3’ ends during the FX reaction, making the DNA fragments ready for adapter ligation. Following this step, Illumina platform-specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding dual bar-coded libraries to a flow cell for sequencing.

Following adapter ligation, the reaction is purified, and any adapter-dimers are removed. This can be automated on various high-throughput automation platforms.

Whole SARS-CoV-2 genome coverage

Based on primer sequences from the ARTIC Network and this publication, the QIAseq SARS-CoV-2 Primer Panel separates 400 bp amplicons into two PCR pools that together cover the entire SARS-CoV-2 genome. Using a QIAseq FX DNA Library UDI Kit, the amplicons from the QIAseq SARS-CoV-2 Primer Panel are brought within the length requirements to perform sequencing on Illumina platforms.

The QIAseq SARS-CoV-2 Primer Panel can be used to generate whole genome coverage of the SARS-CoV-2 viral genome for epidemiological research purposes.

With the ability to multiplex up to 384 samples, the panel allows labs to rapidly scale up sequencing capabilities and ramp up SARS-CoV-2 genomic surveillance efforts. Accurate amplification and high SARS-CoV-2 genome coverage enables the detection of new variants with unparalleled precision.