Scale up your NGS library prep
for SARS-CoV-2 research and detection

“Tecan's Trio RNA-Seq Library Preparation Kit has successfully delivered high-quality results for viral sequencing of nasal swab samples for our COVID-19 research. It has proven to be the right approach to meet the increasing need for a solution in these exceptional times.”

DreamPrep™ NGS workstation is a complete solution that automates NGS library preparation kits for consistent, high-throughput sample-to-sequence libraries for detection, surveillance and evolution studies in your lab.

The automated workflow for the Trio RNA-Seq™ Library Preparation Kit on DreamPrep NGS is available now.

Results of Trio RNA-Seq automated on the Fluent Liquid Handler integrated with Infinite plate reader and Inheco On-Deck Thermal Cycler - Final library yields of 24 low-input samples (500pg, UHR) prepared using Trio RNA-Seq demonstrates consistency with a 10.9% CV across replicates, with no library failures. The workflow shows a significant depletion of unwanted human rRNA which aids in enriching sequences of interest.

Trio RNA-Seq incorporates proprietary technologies that generate RNA-Seq libraries from precious, low-input samples, while depleting abundant uninformative sequences. These powerful technologies include:

  • Single Primer Isothermal Amplification (SPIA) enables access to limited and degraded samples as low as 500 pg. SPIA increases detection sensitivity, effectively addressing the challenge of low input and low viral titer, particularly in asymptomatic patients or healthy carriers
  • Enzymatic fragmentation and DimerFree library construction allows efficient and robust library preparation
  • AnyDeplete removes abundant uninformative sequences, such as human ribosomal RNA (rRNA), to maximize detection sensitivity without excessive sequencing

Is low abundance of the virus in challenging samples a real issue? Do you want to evaluate how the virus mutates over time? Are you interested in assessing how the virus impacts the human transcriptome during infection?

Our Trio RNA-Seq library preparation kit helps you answer all these questions. The method increases detection sensitivity from nasal swab samples, and is the method of choice for IGATech. (Data has been generated and analyzed by IGATech in the framework of a research initiative coordinated by Istituto di Genomica Applicata and funded by ARGO (Italy)).

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Table 1: Total RNA for NGS library preparation has been extracted using Siemens VERSANT kPCR from 10 nasal swabs ( One sample that tested negative for SARS-Cov-2 used as negative control) and two SARS CoV-2 virus isolates as positive controls. Libraries were prepared using Trio RNA-Seq and sequenced on Illumina Novaseq 2x150bp. The sequencing results demostrate high sequence coverage for SARS-CoV-2 from ultra low input samples, indicating high sensitivity of the Trio RNA-Seq workflow from low abundant viral samples. Complete viral sequence coverage can be achieved with 100M reads for Ct<27, 50M reads for Ct<25 and 10M reads for Ct<22. The final library yield is consistent (with an average of 28 nM) across the different input amounts showing the robustness of the SPIA amplification combined with the library preparation, making it suitable for processing samples of widely varying inputs and viral load.

Graph 1: The average read distribution across the nasal swab samples shows that there is high bacterial background along with host (human) seqeunces. The Trio RNA-Seq library prep comes integrated with uninformative transcript depletion to enable enrichment of viral sequence of interest. Data have been normalized removing the unaligned reads.

 

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Recent peer-reviewed studies (published in 2020) demonstrate the successful application of the Trio RNA-Seq Library Preparation Kit in detecting the novel coronavirus in patient samples:

  • Maurano, MT., et. al. from New York University successfully performed large scale genomic surveillance of 236 patient samples from the NYC metropolitan area. The researchers showed that there was undetected community spread and concluded that the majority of SARS-CoV-2 strains in NYC originated from Europe. Trio RNA-Seq was chosen over other kits for this study due to a lower rate of duplicate reads, especially for lower viral input samples.
  • Shen, Z., et. al. performed metatranscriptome sequencing for the bronchoalveolar lavage fluid of eight SARS-CoV-2 patients, 25 community-acquired pneumonia (CAP) patients and 20 healthy controls to study viral evolution and how the virus interacts with other microorganisms in the lung . The authors concluded that the virus evolves after infection, which may impact its virulence, infectivity, and transmissibility.
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