Understanding Coronavirus with PacBio sequencing
As a leading provider of long-read sequencing technology, PacBio is committed to supporting the efforts of government, academic, and commercial labs in their efforts to understand SARS-CoV-2 and COVID-19.
Learn how you can use HiFi reads to enable your viral research, from understanding viral genomes to the host immune response.
Below are resources to aid scientists in the study of SARS-CoV-2 and the related immune response to COVID-19
With long-read amplicon sequencing you can gain access to the viral RNA genome
How Can PacBio Highly Accurate Long-read Sequencing Help with Viral Sequencing?
- HiFi reads provide high accuracy (>99%) for full-length amplicon sequencing with multiplexing
- By facilitating SNP phasing and quasispecies resolution, giving insights into viral evolution within a host, over time within a community, or across geographic regions
- Through detection of rare variants linked to immune evasion or drug resistance
Whole Genome Tiled Amplification Protocols:
- PacBio Compatible Mt. Sinai Protocol for SARS-CoV-2 Sequencing;~1.5 or ~2.0 kb tiled amplicons
- Multiplexing 2.5 kb Amplicons for Whole Genome Sequencing of SARS-CoV-2
- Hiscox NIHR HPRU / PHE protocol; ~1 kb tiled amplicons
- PacBio Compatible CDC Protocols for SARS-CoV-2 Sequencing; ~500 or ~900 bp tiled amplicons
- Freed protocol for amplification of SARS-CoV-2; ~1.2 kb tiled amplicons
- Amplification of Full-Length SARS-CoV-2 Spike Gene with Barcoded Primers
- End-to-end method for inexpensive, high-throughput COVID-19 screening with RT-PCR and sequencing validation
- VAST/Pasteur Method for SMRT-Sequencing of SARS-CoV-2 from Cultured Virus Supernatant
- Recommended barcoding method: PacBio Barcoded Overhang Adapters
Options for Barcoding
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded Overhang Adapters for Multiplexing Amplicons for multiplexing a limited number of samples
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded M13 Primers for Multiplex SMRT Sequencing for multiplexing up to 1,024 samples
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded Universal Primers for Multiplexing Amplicons for multiplexing up to 96 samples with the PacBio-supplied 96 Barcoded F/R Universal Primers Plate
Assay Development Resources
- Global Initiative on Sharing All Influenza Database (GISAID; strain ID EPI_ISL_405839) SARS-CoV-2 sequences
- Twist synthetic SARS-CoV-2 controls and research panels
- ATCC SARS-CoV-2 resources
- Starr, N. T. et al., (2020) Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding. bioRxiv Preprint.
- Djiking, A., and Spiro, D. (2009) Advancing full length genome sequencing for human RNA viral pathogens. Future Virology. 4(1): 47–53.
- Laird Smith, M., et al. (2016) Rapid sequencing of complete env genes from primary HIV-1 samples. Virus Evolution, 2(2), vew018.
- Betz-Stablein, B. D., et al. (2016) Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation. Journal of Virology, 90, 7171–7183.
- Brese, R. L., et al. (2018) Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues. Journal of Neurovirology, 24(4), 439–453.
- Tao, Y., et al. (2017) Surveillance of bat coronaviruses in Kenya identifies relatives of human coronaviruses NL63 and 229E and their recombination history. Journal of Virology, 91(5), e01953–16.
- Lu, X., et al. (2017) Spike gene deletion quasispecies in serum of patient with acute MERS-CoV infection. Journal of Medical Virology, 89(3), 542–545.
- Le Nouën, C., et al. (2017) Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure. PNAS, 114(3), E386–E395.
- Li, Y., et al (2020) Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions. Virus Genes.
Understanding the Immune Response to COVID-19
With long reads you can sequence full-length B Cell Receptor (BCR) Repertoire and the IGH locus
How Can PacBio Highly Accurate Long-read Sequencing Help with Understanding Immune Function?
- Full-length BCR sequencing allows priming from the most conserved region of the constant domain, yielding isotype information while reducing both genetic bias from variants that impact primer binding and PCR bias arising from highly multiplexed amplification reactions, all without any assembly.
- Full-length BCR sequencing allows detection of all variants resulting from VDJ recombination and affinity maturation, including those outside the CDR3 region that arise during hypermutation.
- Capture and assemble the ‘hard to sequence’ IGH region to understand how allelic variation in immune genes correlates with patient outcomes.
- ARTISAN PCR Supplementary materials, with protocol and primers
- Procedure & Checklist – Preparing SMRTbell Libraries using PacBio Barcoded Overhang Adapters for Multiplexing Amplicons
- Roe, D., et al. (2020) Efficient sequencing, assembly, and annotation of human KIR haplotypes. bioRxiv Preprint.
- Gilchuk, P., et al. (2020) Analysis of a therapeutic antibody cocktail reveals determinants for cooperative and broad Ebolavirus meutralization. Immunity, 53(2), 388-403.e12.
- A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus. bioRxiv Preprint. ,
- Lucas, J. A. M., et al. (2020) Single Molecule Real‐Time DNA sequencing of the full HLA‐E gene for 212 reference cell lines. HLA.
- Mincarelli, L., et al. (2020) Combined single-cell gene and isoform expression analysis in haematopoietic stem and progenitor cells. bioRxiv Preprint.
- Koning, M. T., et al. (2016) ARTISAN PCR: rapid identification of full-length immunoglobulin rearrangements without primer binding bias. British Journal of Haematology, 178, 979–994.
- DeKosky, B. J., et al. (2015) In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire. Nature Medicine, 21(1), 86–91.
- Watson, C. T., et. al. (2013) Complete Haplotype Sequence of the Human Immunoglobulin Heavy-Chain Variable, Diversity, and Joining Genes and Characterization of Allelic and Copy-Number Variation. Am J Hum Genet. 92(4): 530–546.
- Watson, C. T., et al. (2019) A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains. Immunology and Cell Biology, 97 (10), 888-901.
- Ford, M., et al (2020) Genotyping and Copy Number Analysis of Immunoglobin Heavy Chain Variable Genes Using Long Reads. iScience 23(3), 100883.
- Mayor, N. P., et. al. (2019) Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study. Biol of Blood and Marrow Transplantation. 25(3):443-450.
Webinar: Understanding SARS-CoV-2 and host immune response to COVID-19 with PacBio sequencing
Connect with a PacBio Scientist
If you are working on coronavirus or COVID-19 and would like to learn more about protocols or service providers, please contact us.