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xGen™ SARS-CoV-2 Midnight Amplicon Panel

Multiplex PCR primers for amplicon sequencing

Panel for amplification of SARS-CoV-2 from research samples that can be used to understand the spread of the virus, identify variants and provide knowledge to enable a better understanding of COVID-19.

xGen NGS—made for COVID-19 research.

Ordering

  • Amplifies the SARS-CoV-2 genome for sequencing using premixed multiplex PCR primers
  • Provides data for understanding SARS-CoV-2 viral evolution and the epidemiology of COVID-19
  • Data identifies SARS-CoV-2 variants, including alpha, beta, gamma, delta, and mu
  • Panel generates 29 tiled amplicons, approximately 1200 bp in length
  • Panel is optimized for high throughput applications or automated workflows
  • Samples with Ct values <32 are recommended for complete genome sequencing coverage based on initial testing

Product details

The Midnight Panel was created by Drs. Nikki Freed and Olin Silander of Massey University in New Zealand [1]. This panel consists of 29 amplicons of approximately 1200 bp in length (Figure 1). The reduced number of amplicons allows for more consistent sequencing across the genome than would be achievable with a higher number of amplicons.

Figure 1. xGen SARS-CoV-2 Midnight Amplicon Panel amplicons. The multiplex primer panel consists of two pools. Pool 1 includes 30 primer pairs that amplify the odd regions of the SARS-CoV-2 genome, and Pool 2 includes 28 primer pairs that amplify the even regions. The spacing is designed to tile across the entire SARS-CoV-2 genome, generating 29 amplicons approximately 1200 bp in length.

Since the primers only anneal to a total of 4.5% of the viral genome, the chance that viral mutations will disrupt primer annealing is lower. This results in a lowered chance of amplicon dropouts.

The Ct value for the amplicon sequencing reaction is an important metric since the higher the Ct, the lower the number of viral genomes in the sample. Samples with less viral genomic material pose difficulty for primer annealing and amplification.

In our experiment, contrived samples were prepared by mixing inactivated SARS-CoV-2 viral material into a negative background matrix of a negative nasopharyngeal (NP) swab in viral transport medium (VTM), with two samples per Ct value. Results show that when the xGen SARS-CoV-2 Midnight Amplicon Panel and xGen Lotus DNA Library Prep Kit are combined, the recommended Ct value is <32 which will ensure that 97% of the SARS-CoV-2 genome will be sequenced with sufficient depth to identify variants (Table 1).

Table 1. Midnight panel and xGen Lotus DNA Library Prep Kit metrics.

Recommended Ct value Coverage (%) Contiguous amplicons (count) Coordinates covered
<32 97% 29 30–28,985

Workflow

Since the length of the amplicons created by the xGen SARS-CoV-2 Midnight 1200 Amplicon Panel are 1200 bp, Illumina® sequencing platforms are not compatible. To overcome this restriction, the amplicons require fragmentation before library construction. By using the IDT xGen DNA Library Prep Kit with enzymatic DNA fragmentation, the xGen SARS-CoV-2 Midnight Amplicon Panel amplicons can then be decoded on Illumina sequencing systems. An overview of this workflow shows the steps necessary for library construction (Figure 2). IDT offers many of the reagents for the workflow, from the SARS-CoV-2 Midnight Panel and the components for library construction, to the Nuclease-Free Water. 

Figure 2. Expected experimental workflow for SARS-CoV-2 genome sequencing using the Midnight Panel in combination with the xGen DNA Library Prep Kit. By using the IDT xGen DNA Library Prep Kit after the xGen SARS-CoV-2 Midnight Amplicon Panel, the original 1200 bp fragments are converted into a library that is compatible with Illumina sequencers and chemistry.

For labs with access to Oxford Nanopore Sequencing instruments, the amplicons created using the xGen SARS-CoV-2 Midnight Amplicon Panel can be prepared using one of the barcoding kits available directly from Oxford Nanopore (e.g., Rapid Barcoding Kit, SQK-RBK004).

Product data

As previously noted, the Ct value for the viral RNA in the sample is a critical metric to obtain usable data. IDT researchers used the xGen SARS-CoV-2 Midnight Amplicon Panel in combination with the IDT xGen DNA Lotus Library Prep Kit on contrived samples that mixed negative nasopharyngeal swab sample with inactivated viral material.

In a proof-of-concept test, sequencing libraries were first prepared according to the xGen SARS-CoV-2 Midnight Amplicon Panel protocol for samples with varying Ct values. These libraries were then subjected to enzymatic fragmentation and library preparation according to the xGen™ Lotus DNA Library Prep Kit recommendations (Figure 3).

Figure 3. Amplicon sequencing combining the xGen SARS-CoV-2 Midnight Amplicon Panel and the xGen Lotus DNA Library Prep provided >99.5% coverage at >5X of the SARS-CoV-2 genome for Ct values <32. Data was generated from two replicates per Ct value with the mean values shown here. Inactivated SARS-CoV-2 viral material in a negative background matrix of a negative nasopharyngeal (NP) swab was collected from healthy donors with three different Ct values and tested: 29.3, 31.4, and 33.2. Libraries were generated in duplicate following the xGen DNA Library Prep Kit recommendations. The resulting libraries was sequenced with MiSeqTM 300 cycle kit (2x151 reads) (Illumina). For Ct 29.3, there were on average 1,714,799 total reads, and 1,710,462 reads that mapped to the human or viral genome reference sequence (99.7%). For Ct 31.4, there were on average 1,105,540 total reads and 1,102,467 reads that mapped to the human or viral genome (99.7%). For Ct value of 33.2, there were on average 1,033,016 total reads and 1,028,369 reads that mapped to the human or viral genome (99.6%). The percentage of reads greater than 5X, 15X, and 30X are shown for each of the libraries. For Ct values less than 32, high coverage of the SARS-CoV-2 genome is evident.

Resources

References

  1. Freed NE, Vlková M, et al. (2020) Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200bp tiled amplicons and Oxford Nanopore Rapid Barcoding. Biol Methods Protoc 5(1):bpaa014.
  2. Freed N and Silander O (2020) nCoV-2019 sequencing protocol (RAPID barcoding, 1200 bp amplicon) v4 (protocols.io.bh7hj9j6). protocols.io.

*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.

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