Australia: Rapid genome strategy to track coronavirus

Australia: Rapid genome strategy to track coronavirus

Australia: Rapid genome strategy to track coronavirus.  Nature Communications | Australian researchers develop rapid genome strategy to track coronavirus.

Thanks to cutting-edge “nanopore” genome sequencing technology, researchers at the Gavin Institute of Medical Research and the Kirby Institute at the University of New South Wales in Sydney have developed the fastest coronavirus genome sequencing strategy in Australia to date. Technological advances may provide critical and timely clues to the association between SARS-CoV-2 infection cases.

Every time the SARS-CoV-2 virus spreads from person to person, replication errors may occur, which may change several of its 30,000 genetic letters. By identifying this genetic variation, it is possible to identify different cases of the coronavirus and know where it might have spread from. Genome testing is essential for tracking the spread of the virus, and if you only investigate known epidemiological contacts, you still cannot know its source.

By reconstructing the evolutionary history or “family tree” of the virus, it can help to understand the spread of COVID-19 and identify the so-called “super spreaders”. Dr. Ira Deveson, head of the genome technology group at the Garvan Kinghorn Clinical Genomics Center and the corresponding author of the original text, said: “When a new’mysterious’ coronavirus case is discovered, every minute is important. At Garvan, we reuse With the genome sequencing function, the coronavirus genome can be quickly analyzed in just a few hours.”

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Pioneering rapid genomics. It is vital to quickly identify the spread of SARS-CoV-2. New South Wales Health Pathology has collaborated with the Garvan Institute and the Kirby Institute to develop a faster SARS-CoV-2 genome sequencing function, which potentially enhances the ability to take quick action to isolate and monitor potential contacts when exposed to tracers ability. Garvan researchers have optimized and debugged the most advanced Oxford nanopore technology experimental method, which can sequence SARS-CoV-2 in less than four hours. Garvan’s Kinghorn Clinical Genomics Center is the first facility in Australia to establish and apply this nanopore technology for SARS-CoV-2 genome monitoring.

High-precision emerging technology. The current gold standard method can only read short gene sequences of 100-150 genetic letters at a time, while nanopore technology has no upper limit on the length of DNA fragments that can be sequenced, and can quickly determine a complete viral genome sequence. However, like many emerging technologies, people are also concerned about the accuracy of nanopore sequencing.

In the paper, the author solved these problems, and reported the results of strict analysis and evaluation of the coronavirus genome sequencing program. The researcher’s analysis shows that the nanopore sequencing method has high accuracy (sensitivity of variants detected in 157 SARS-CoV-2 positive patient specimens> 99%, accuracy> 99%), and provides the most The researchers hope that these guidelines will promote the adoption of the technology by other teams around the world.

The researchers say that nanopore sequencing can even enhance SARS-CoV-2 surveillance by enabling point-of-care sequencing and shortening the turnaround time of critical cases. Nanopore devices are cheaper, faster, and more portable, and do not require the laboratory infrastructure required by current standard pathogen genomics tools.

Variation detection performance of SARS-CoV-2 whole genome ONT sequencing

By performing ONT sequencing on matched SARS-CoV-2 specimens, the sensitivity of Illumina to SNV (n = 157) was detected at the same level of variation frequency (80-100%). Figure 1C shows the correlation of the observed mutation frequency of SNV candidates detected at sub-consensus frequencies (20-80%) using Illumina and ONT sequencing.

Candidates detected with ONT but not Illumina are considered false positives (FP; red), and candidates detected with Illumina but not detected ONTs are considered false negatives (FP; pink). All in all, ONT sequencing can achieve high-precision and reproducible detection of SNV at a common level among SARS-CoV-2 patient isolates, but it is generally not suitable for detecting small indel variants.

Using Varscan2, 154 sub-consensus SNV candidate genes were identified in the ONT sequencing library. 119 SNVs (sensitivity = 76.3%) and 25 false positives (accuracy = 82.6%) were detected in the Illumina comparison set. The read count frequencies of the variants identified by these two techniques are correlated (R2 = 0.69), indicating that they are real variants, not artificial (Figure 1c).

Although the overall performance of subconsensus SNV testing is quite poor, most false positives and false negatives are limited to the lower limit of the frequency range evaluated here (Figure 1c, d). For example, with high sensitivity (95.7%, 91.3%) and accuracy (100%, 97.7%), SNVs with high (60–80%) and medium (40–60%) consensus frequencies are detected. Frequency variation (20-40%) has low detection sensitivity (63.2%) and low accuracy (69.6%; Figure 1d). Not surprisingly, the indel error rate in ONT sequencing libraries is high, which means they are not suitable for detecting indel diversity.

All in all, ONT sequencing can perform highly accurate and reproducible detection of common levels of SNV in SARS-CoV-2 patient isolates, but it is generally not suitable for detecting small indel variants.

Sum up

Due to the relatively low mutation rate observed in SARS-CoV-226, accurate sequence determination is essential to correctly define the phylogenetic structure of the disease outbreak. It is known that ONT sequencing has a higher read-level sequencing error rate than short-read technology, and there is reasonable concern about the applicability of this technology to the SARS-CoV-2 genome. In addition, public databases used for SARS-CoV-2 data (such as GISAID: https://www.gisaid.org/) already contain consensus genome sequences generated by ONT sequencing, which may confuse research that relies on these resources.

All in all, ONT sequencing can achieve high-precision and reproducible detection of SNV at a common level among SARS-CoV-2 patient isolates, but it is generally not suitable for detecting small indel variants. Although the high error rate of indels in ONT sequencing prevents accurate detection of small indels, the long nanopore readout seems to be very suitable for detecting large deletions and other potential structural variations. This technological advancement proves what can happen when Public Pathology collaborates with the Institute to achieve a common goal.

(source:internet)

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