Since the outbreak of SARS-CoV-2, scientists have identified pathogens at an alarming rate and determined the entire genome of the virus. Because rapid diagnosis is critical to the management and treatment of the disease.
In order to isolate the SARS-CoV-2 ribonucleic acid as soon as possible, on the basis of next-generation sequencing (high-throughput sequencing) technology and provide a useful tool for monitoring and researching SARS-CoV-2, a research team from McMaster University in Canada quickly designed a bait capture platform for the next-generation sequencing of SARS-CoV-2 without culture and amplification. Genes were found in complex samples and verified by samples, which will help prevent the current outbreak.
The new coronavirus is a beta-type coronavirus with an envelope and a diameter of 60-140 nm. Its genetic material is a single RNA strand, close to 30,000 bases in length. It belongs to the same family as SARSr-CoV and MERSr-CoV. At present, the closest is the bat SARS-like coronavirus (bat-SL-CoVZC45), which has more than 85% homology.
The RNA core of the new coronavirus is surrounded by a protein shell, and the surface is highlighted by S protein, which is the key to infect host cells. In Science in February 20, scientists used cryo-electron microscopy to analyze the molecular structure of spike protein and found that it acts like the SARS virus and invades cells by interacting with ACE2 receptors in human cells.
But its affinity with ACE2 is 10-20 times that of SARS virus, which is likely to be the cause of high contagion. And with a high mutation rate (approximately 10-4 nucleotide substitutions per site per year), genome-wide monitoring is essential for proper molecular epidemiology (ie, tracking changes throughout the genome). Although the second-generation PCR technology is currently the main method for monitoring SARS-CoV-2, the application of no amplification and no culture methods to isolate SARS-CoV-2 RNA, together with high-throughput sequencing technology, will provide a more effective monitoring tool for SARS-CoV-2.
High-throughput sequencing technology, also known as “next-generation” sequencing technology, is marked by its ability to perform sequencing and short read lengths on hundreds of thousands to millions of DNA molecules in parallel at a time. Because of its high throughput, independence from known nucleic acid sequences, and higher sensitivity, it has played an important role in the identification, typing, traceability, and diagnosis of new coronaviruses.
Although a probe set for targeted enrichment of multiple viruses already exists, the team proposed a detection device specifically for the current SARS-CoV-2 outbreak. For probe sets based on a wide range of viral diversity, attention needs to be paid to off-target hybridization, leading to off-target concentration and increased workflow time and cost. Considering the importance of off-target hybridization and balancing the length and depth of coverage, The device has improved in detecting rare DNA related to antibiotic resistance. It is designed to maximize specificity and sensitivity.
At present, in order to sequence SARS-CoV-2, a culture step is needed. Without facilities for culturing the virus, it is difficult to analyze SARS-CoV-2. Use a small nanopore sequencer to trap SARS-CoV -2 and then NGS bait capture provides an easier option. Targeted SARS-CoV-2 RNA enrichment facilitates enrichment by physically isolating target RNA from complex patient samples, making most sequenced fragments a target, reducing the need for metagenomic sequencing. It also reduces overall turnaround time and costs, and enables rapid genetic analysis.
By removing candidate probes that may hybridize to humans or other species, the sensitivity to SARS-CoV-2 is enhanced, however, e-karyotic tools cannot accurately identify the RNA or DNA of eukaryotes, bacteria, or archaea. In the case of reflection hybridization in solution, the probe set proposed by the research team is likely to hybridize with other coronaviruses (such as SARS-CoV and MERS-CoV). This is the current shortcoming of the probe device. In the past, members of the team have used bait capture isolation and sequence analysis for the cause of black death, and recently designed and validated an accurate and sensitive enrichment platform for NGS detection. The research team also stated that they will continue their efforts to release the formal technology for SARS-CoV-2 research as soon as possible.
CD Genomics was established in 2004, the company aims at providing the research community with high quality Next Generation Sequencing, PacBio SMRT sequencing, and microarray services. Due to the demand of services has being increased, CD Genomics has already updated its technology platform to mainstream NGS and microarray instruments. At present, its senior bioinformaticians have ever viewed more than ten thousands of trace files and accumulated abundant experience with our Illumina HiSeq 2500, HiSeq 4000, Miseq Benchtop Sequencer, PacBio Sequel, PacBio RS II, Ion Torrent PGM, and ABI 3730/3730XL analyzer, etc. The company continues to work hard to offer the same dependable services to pharmaceutical and biotech companies, as well as academia and government agencies for the purpose of satisfying all sequencing or array needs.