Collibri NGS Advancements for Viral Surveillance

Understanding disease transmission pathways requires collaboration between governmental and private organizations. Government-sponsored programs, such as the Global Influenza Programme (GIP) [1] that is sponsored by the World Health Organization (WHO), establish standards and offer training.

Public-private consortiums, such as the academic labs that partner with the Center for Disease Control (CDC) in the United States, collaborate on viral surveillance for pandemics such as SARS-CoV-2 (COVID-19) to collect and analyze viral samples around the world. This allows the scientific community to monitor global and local trends in virus transmission and to support the selection of strains for vaccine production. Data on the rate of viral transmission may also inform government decisions on transportation, health screening checks, and potential quarantine enforcement policies.

Non-governmental groups such as Nextstrain.org assist with viral surveillance by compiling public databases and visualizing trends in viral transmission. NGS surveillance can reveal cryptic transmission patterns in which a pathogen is undetected by non-NGS surveillance programs. By sequencing 346 SARS-CoV-2 viruses collected in Washington State prior to mid-March of 2020, including samples obtained from the Seattle Flu Study, Hodcroft and colleagues from the United States and Switzerland estimated that the virus was transmitting undetected in Washington State during January and February of 2020 [2].

While the number of available full genomes produced by NGS can grow rapidly, datasets have historically faced challenges in the quality of the sequences. High-quality sequences are required to trace epidemics, monitor for potentially emerging viruses within the population, develop potential anti-viral therapies, identify targets for vaccine development, and understand host-pathogen interactions.

One option to improve the quality of sequences is to use library preparation kits that are designed to handle a wide variety of sample qualities and genome sizes to produce consistent, full-genome coverage. In comparison to transposomic methods, the Invitrogen Collibri DNA Library Prep Kits for Illumina Systems consistently provide full coverage of pathogen and host genomes. The Collibri DNA kits are suitable for genomes of all sizes and may be used to sequence both the pathogen and the host to perform host-pathogen studies.

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To improve accuracy of RNA sequencing results for RNA viruses and other samples, the Invitrogen Collibri Stranded RNA Library Prep Kits for Illumina Systems use a unique protocol in which helper adapters are added directly to RNA. The helper adapters are extended into full-length adapters for Illumina NGS systems during PCR in the final steps of the workflow. Unlike library prep kits that convert RNA into cDNA prior to addition of adapters, diversity of RNA transcripts is retained by the Collibri library prep kits because cDNA priming is not affected by GC content. In addition, ligation of helper adapters directly to RNA enhances accuracy of NGS results because random oligonucleotide sequences are not incorporated into cDNA, preventing false SNPs and point mutations.

The Collibri Stranded RNA protocol is rapid (4.5 hours) and produces highly stranded results, enabling detection of overlapping genes. The protocol is suitable for RNA sequencing of genomes of all sizes and so is suitable to study viral genomes, host gene expression, and host-pathogen interactions.

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	Collibri DNA Library Prep kits	Collibri Stranded RNA Library Prep kits**
NGS method	Whole-genome sequencing	sequencing
Viral insights	de novo assembly of novel pathogens Genomic mutations Transmission pathways Research host susceptibility to infection and predict interferon response	Detect coding RNA Gene expression Gene fusions Transcript isoforms
Suitable for degraded RNA	Yes after reverse transcription*	Yes
Workflow time	1.5 hr***	4.5 hr
Compatible with all Illumina NGS systems	Yes	Yes

For accurate quantification of libraries prior to sequencing, we recommend using the Collibri Library Quantification Kit or Qubit assays.
* SuperScript IV Reverse Transcriptase is recommended to perform reverse transcription
** The Dynabeads DIRECT Purification Kit is recommended to perform enrichment prior to library preparation.
*** Optional PCR adds up to one hour

1. Global Influenza Programme
2. Bedford T, et al. Cryptic transmission of SARS-CoV-2 in Washington State. medRxiv 2020.04.02.20051417; doi: https://doi.org/10.1101/2020.04.02.20051417.
3. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, Sagulenko P, Bedford T, Neher RA. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018 Dec 1;34(23):4121-4123.