QIAseq DIRECT SARS-CoV-2 Kits

QIAseq DIRECT SARS-CoV-2 Kits are specially designed to advance research into SARS-CoV-2, which is the causative agent of COVID-19 disease. These kits are single-box solutions for sequencing the entire viral genome. SARS-CoV-2 is encoded by a positive-sense, single-stranded RNA molecule that can be mixed with host RNA during isolation from a sample. The kits include reagents to reverse transcribe the RNA into cDNA, primers to specifically enrich for the viral genome and Unique Dual Indices (UDIs) for sequencing. The panel consists of approximately 550 primers for creating 425 amplicons, covering the entire SARS-CoV-2 viral genome.
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High-quality, single-box solution

Target amplification of SARS-CoV-2 requires both reverse transcription and whole genome enrichment of the viral RNA. The QIAseq DIRECT SARS-COV-2 Kit is a single-box solution for generating whole genome libraries that are sequencing-ready and compatible with all Illumina platforms.

Novel primer design

QIAseq DIRECT SARS-CoV-2 Kit is designed for optimal performance on short-read sequencing platforms. It generates amplicons that are approximately 250 bp, compared to 400 bp amplicons produced by alternative methods, resulting in shorter amplicons that don't require fragmentation. The kit generates high-yield, similar-sized libraries that lead to performance improvements in sequence uniformity and overall read depth.

Multiplexing 

The QIAseq DIRECT SARS-CoV-2 Kit enables multiplexing on high-throughput Illumina instruments, such as the NovaSeq (up to 384 samples per lane of an Illumina flow cell).

Data analysis 

When paired with QIAGEN CLC Genomics Workbench, the panel delivers data that can be quickly analyzed. Sequence data can be used to identify variants across different samples, as well as to compare with 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 a 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 viral genome.

Although next-generation sequencing (NGS) has become a vital tool, library preparation remains a key bottleneck in the NGS workflow. The QIAseq DIRECT SARS-CoV-2 Kit is a multiplex PCR primer set for whole genome amplification of SARS-CoV-2. Using amplicons optimized for short-read platforms, the QIAseq DIRECT SARS-CoV-2 Kit uses an overlapping and redundant targeting approach to reduce the risk of drop outs caused by novel viral mutations.

QIAseq DIRECT SARS-CoV-2 utilizes a streamlined, 4-hour workflow for enrichment and library prep of the SARS-CoV-2 virus genome.

cDNA synthesis and SARS-CoV-2 enrichment

The QIAseq DIRECT SARS-CoV-2 workflow begins with random-primed cDNA synthesis (no rRNA depletion or poly-A selection required). This reaction is flexible with regard to input RNA; 5 µl viral RNA input is required as a starting volume, regardless of viral titer. Following cDNA synthesis, multiplexed primer pools are used in a high-fidelity multiplex PCR reaction to prepare two pools of approximately 250 bp amplicons. The two enriched pools per sample are then combined into a single tube and purified using a QIAseq Bead cleanup step.

Library amplification and sample indexing

Following quantification and normalization, SARS-CoV-2 enriched samples are amplified and sample-indexed using a high-fidelity amplification reaction. During this reaction, Unique Dual Indices (UDIs) are added to the samples. UDIs effectively mitigate the risk of read mis-assignment due to index hopping. This is enabled by filtering mis-assigned reads during the demultiplexing of individual samples, thus generating highly accurate output data. For more information on QIAseq UDIs, please refer to "Appendix A: QIAseq DIRECT Unique Dual Indexes" in the QIAseq DIRECT SARS-CoV-2 Handbook.

Next-generation sequencing 

QIAseq DIRECT SARS-CoV-2 libraries are compatible with Illumina NGS platforms including iSeq 100, MiniSeq, MiSeq, NextSeq 500/550, HiSeq 2500, HiSeq 3000/4000 and NovaSeq 6000. 

The QIAseq DIRECT SARS-CoV-2 Kit can be used to achieve whole genome coverage of the SARS-CoV-2 viral genome for epidemiological research and genomic surveillance 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.