SARS-CoV-2 Lineage Variant Summary

Primers that do not overlap with variants associated with these lineages are shown in blue below a schematic SARS-CoV-2 genome. Primers that overlap with enough sufficiently-frequent variants to be potentially impacted (see below) are indicated in orange. Relevant variants are shown in gray directly below the genome, above the primer sets.

The score listed for a variant is the percent of sequences it occurs in for the selected lineage set. The score listed for a primer is determined by the scoring algorithm described below.

Lineage:

Show primer sets:

{"primer_set_0":["Broughton_N","Broughton N"],"primer_set_1":["CDC_Flu_SC2","CDC Flu/SC2"],"primer_set_2":["Charite_Hospital","Charité Hospital"],"primer_set_3":["China_CDC","China CDC"],"primer_set_4":["Covid_Detective","Covid Detective"],"primer_set_5":["Dudley_As1e","Dudley As1e"],"primer_set_6":["Hong_Kong_University","Hong Kong University"],"primer_set_7":["IP2","IP2"],"primer_set_8":["Japan_NIID","Japan NIID"],"primer_set_9":["LCA_N2","LCA_N2"],"primer_set_10":["Lamb_Orf1A","Lamb Orf1A"],"primer_set_11":["Mason_N1","Mason N1"],"primer_set_12":["Mason_iCare","Mason/iCare"],"primer_set_13":["MasonApr22","MasonApr22"],"primer_set_14":["MasonMar2022","MasonMar2022"],"primer_set_15":["NEB_LAMP_(N2+E1)","NEB LAMP (N2+E1)"],"primer_set_16":["NEB_Luna_qPCR_CDC","NEB Luna qPCR/CDC"],"primer_set_17":["OmniSARS2","OmniSARS2"],"primer_set_18":["RGN","RGN"],"primer_set_19":["Saliva_Direct_CDC_N1","Saliva Direct/CDC N1"],"primer_set_20":["Thailand_NIH","Thailand NIH"],"primer_set_21":["UtrechtU","UtrechtU"],"primer_set_22":["Wehi_S","Wehi_S"]}

{"primer_set_0":false,"primer_set_1":false,"primer_set_2":false,"primer_set_3":false,"primer_set_4":false,"primer_set_5":false,"primer_set_6":false,"primer_set_7":false,"primer_set_8":false,"primer_set_9":false,"primer_set_10":false,"primer_set_11":false,"primer_set_12":false,"primer_set_13":false,"primer_set_14":false,"primer_set_15":true,"primer_set_16":true,"primer_set_17":false,"primer_set_18":false,"primer_set_19":false,"primer_set_20":false,"primer_set_21":false,"primer_set_22":false}

{"data_server":"https://primer-monitor.neb.com/igvstatic","organism_taxid":2697049,"organism_name":"SARS-CoV-2","reference_accession":"NC_045512.2"}

{"lineage_set_0":["A","A.*"],"lineage_set_1":["B","B.*"],"lineage_set_2":["BA.2.86","BA.2.86.*"],"lineage_set_3":["BD.1","BD.1.*"],"lineage_set_4":["BD","BD.*"],"lineage_set_5":["BF","BF.*"],"lineage_set_6":["BJ.1","BJ.1.*"],"lineage_set_7":["BJ","BJ.*"],"lineage_set_8":["BM","BM.*"],"lineage_set_9":["BQ.1.1","BQ.1.1.*"],"lineage_set_10":["BQ.1","BQ.1.*"],"lineage_set_11":["BQ","BQ.*"],"lineage_set_12":["CH","CH.*"],"lineage_set_13":["EG.5.1.1","EG.5.1.1.*"],"lineage_set_14":["EG.5.1.3","EG.5.1.3.*"],"lineage_set_15":["EG.5.1.6","EG.5.1.6.*"],"lineage_set_16":["EG.5.1","EG.5.1.*"],"lineage_set_17":["EG.5","EG.5.*"],"lineage_set_18":["EG.6","EG.6.*"],"lineage_set_19":["EG","EG.*"],"lineage_set_20":["FG.3","FG.3.*"],"lineage_set_21":["FG","FG.*"],"lineage_set_22":["FL","FL.*"],"lineage_set_23":["GE.1","GE.1.*"],"lineage_set_24":["GE","GE.*"],"lineage_set_25":["GJ","GJ.*"],"lineage_set_26":["XBB.1.16.11","XBB.1.16.11.*"],"lineage_set_27":["XBB.1.16.15","XBB.1.16.15.*"],"lineage_set_28":["XBB.1.16.6","XBB.1.16.6.*"],"lineage_set_29":["XBB.1.16","XBB.1.16.*"],"lineage_set_30":["XBB.1.18","XBB.1.18.*"],"lineage_set_31":["XBB.1.28","XBB.1.28.*"],"lineage_set_32":["XBB.1.5.47","XBB.1.5.47.*"],"lineage_set_33":["XBB.1.5","XBB.1.5.*"],"lineage_set_34":["XBB.1.9.1","XBB.1.9.1.*"],"lineage_set_35":["XBB.1.9.2","XBB.1.9.2.*"],"lineage_set_36":["XBB.1.9","XBB.1.9.*"],"lineage_set_37":["XBB.1","XBB.1.*"],"lineage_set_38":["XBB","XBB.*"],"lineage_set_39":["XR","XR.*"],"lineage_set_40":["all","All"]}

Calculating variant impact:

To distinguish primers that are likely to be actually impacted from ones affected by a rare variant or a sequencing error, impact is determined by both the the number of times each variant appears in the data, and the number, length, and position of variants overlapping a primer. To eliminate sequencing errors and other low-frequency variants that will likely not have a significant impact on primers, a variant is only considered in the calculation if it occurs either in at least 1% of sequences (and no less than 10 sequences) in the dataset (after filtering by lineage). Primers are then scored based on the length and position of overlapping variants, and labeled as affected if this results in a sufficiently high score. To focus on recent mutations in the SARS-CoV-2 virus, only variants from the last 180 days are considered.

The composite score for a primer is the sum of all overlapping variant scores. Variant scores are based on length, whether they are indels or mismatches, and position in the primer. Since long variants are more likely to disrupt primer binding, an exponentially-increasing length penalty is added. To account for variant position and indels, a flat multiplier is applied to the score for the relevant bases. Indels and variants 3-5 nt in from (but not at) the 3' end of a primer have the highest multipliers, as these traits empirically relate primer disruption. Variants ≤3 nt from the 5' end of the primer, or ≤2 nt from the 3' end have multipliers that reduce the score compared to the default for any other position. We consider variants at these positions to be less likely to affect polymerase binding. For 5' variants, this is because it creates a flap on the 5' end, which interferes less with amplification. For variants at the 3' end, we assume that 3’-5’ exonuclease activity in commonly used polymerases can remove mismatched bases. While we attempt to model the observed effects of variants observed, it is likely imperfect. Proposed variant impacts should be assessed experimentally as we did for early omicron, BQ.1 associated variation and more generally for LAMP applications

Overlapping amplicons:

The NEB LAMP E2019 E gene amplicon overlaps with a variant observed in the BA.1, BA.1.1, and BA.2 (omicron) lineages.
The CDC/NEB qPCR E3019 N1 gene probe overlaps with a variant observed in the BA.1 (omicron) lineage.
The CDC/NEB qPCR E3019 N1 gene R primer overlaps with a variant observed in Delta lineages.
The US CDC/Flu SC2 N Gene probe overlaps with a variant observed in the Delta and BA.2 lineages.
The China CDC N gene qPCR amplicon overlaps with variants in all lineages.
The Charité RdRp primers overlap with variants observed in Delta and BA.1 lineages
The Charité E amplicon overlaps with a variant observed in BA.1, BA.1.1, and BA.2 (omicron) lineages.
The Multiplex amplicon designs (ARTIC v3, v4, VarSkip, Midnight 1200 bp, ...) overlap with multiple variants:

Methods:

Sequences reported in Sequence Sources for each lineage were aligned to the NC_045512.2 reference sequence (minimap2 -r 10000 --score-N=0). Variants > 2% frequency (freebayes) were evaluated for overlap with primer regions (bedtools) and displayed (Geneious Prime 2021.0.3). Due to the rapid increase in omicron sequences, BA.1, BA.1.1 and BA.2 lineage variants were identified by classifying recent consensus sequences submitted to GISAID using pangolin --usher before alignment and variant calling (2% frequency threshold). Variants were manually filtered to remove Delta contamination. Exclusion criteria: Variants present in Omicron lineage sequences and Delta lineage sequences that dropped in frequency between November 2021 and January 2022 were excluded.

Sequence Sources:

AY.* (combined delta) This is a composite track containing variants from all circulating AY.lineages as of 2022-02-01. No single virus will contain all these mutations. It includes variants from lineages AY.3, AY.4, AY.25, AY.25.1, AY.43, AY.44, AY.100, AY.103, AY.122

B.1.617.2: GitHub. “Potential Sequences That Should Be Included in B.1.617 · Issue #49 · Cov-Lineages/Pango-Designation.” Accessed June 20, 2021. https://github.com/cov-lineages/pango-designation/issues/49.

AY.3: GitHub. “B.1.617.2 Sub-Lineage with ORF1a: I3731V · Issue #121 · Cov-Lineages/Pango-Designation.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/issues/121.

AY.4: GitHub. “Proposal for Sub-Lineages within B.1.617.2 · Issue #180 · Cov-Lineages/Pango-Designation.” Accessed October 1, 2021. https://github.com/cov-lineages/pango-designation/issues/180.

AY.25: GitHub. “B.1.617.2 Sublineage with Expansion in Cape Cod, Massachusetts, USA in July 2021 · Issue #181 · Cov-Lineages/Pango-Designation.” Accessed October 1, 2021. https://github.com/cov-lineages/pango-designation/issues/181.

AY.25.1: GitHub. “Proposal of New Sub-Lineage of Delta AY.25 for Peru with Principal Mutation in Spike: D796Y · Issue #355 · Cov-Lineages/Pango-Designation.” Accessed February 11, 2022. https://github.com/cov-lineages/pango-designation/issues/355.

AY.43: GitHub. “Proposal for Large European Delta Sub-Lineage with N:9L, ORF1b:829I (~80k Seqs) · Issue #240 · Cov-Lineages/Pango-Designation.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/issues/240.

AY.44: GitHub. “Proposal for Large US Delta Sub-Lineage with Orf1b:183V (~80k Sequences) · Issue #242 · Cov-Lineages/Pango-Designation.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/issues/242.

AY.100: GitHub. “Added New Lineage AY.100 with 129 New Sequence Designations · Cov-Lineages/Pango-Designation@f792f5d.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/commit/0892b9f7e2f19f7f8d9e655de277083747b267e1.

AY.103: GitHub. “Added New Lineage AY.103 with 18135 New Sequence Designations and 13 … · Cov-Lineages/Pango-Designation@0892b9f.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/commit/0892b9f7e2f19f7f8d9e655de277083747b267e1.

AY.122: GitHub. “Still Undesignated Orf1a:K261N Delta Sublineage Is Dominant in Russia and Widespread in Europe · Issue #320 · Cov-Lineages/Pango-Designation.” Accessed February 11, 2022. https://github.com/cov-lineages/pango-designation/issues/320.

BA.1 (omicron): GitHub. “B.1.1 decendant associated with Southern Africa with high number of Spike mutations · Issue #343 · Cov-Lineages/Pango-Designation.” Accessed November 27, 2021. https://github.com/cov-lineages/pango-designation/issues/343.

BA.1.1 (omicron): GitHub. “Omicron Sublineage with Potentially Beneficial Mutation S:346K · Issue #360 · Cov-Lineages/Pango-Designation.” Accessed February 11, 2022. https://github.com/cov-lineages/pango-designation/issues/360.

BA.2 (omicron): GitHub. “Proposal to Split B.1.1.529 to Incorporate a Newly Characterised Sibling Lineage · Issue #361 · Cov-Lineages/Pango-Designation.” Accessed February 11, 2022. https://github.com/cov-lineages/pango-designation/issues/361.

Amplicon Designs:

US CDC: Lu, Xiaoyan, Lijuan Wang, Senthilkumar K. Sakthivel, Brett Whitaker, Janna Murray, Shifaq Kamili, Brian Lynch, et al. “US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 - Volume 26, Number 8—August 2020 - Emerging Infectious Diseases Journal - CDC.” Accessed April 23, 2021. https://doi.org/10.3201/eid2608.201246.

China CDC: “新型冠状病毒核酸检测引物和探针序列（Specific Primers and Probes for Detection 2019 Novel Coronavirus）.” Accessed June 20, 2021. http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html.

Charité: Corman, Victor M., Olfert Landt, Marco Kaiser, Richard Molenkamp, Adam Meijer, Daniel KW Chu, Tobias Bleicker, et al. “Detection of 2019 Novel Coronavirus (2019-NCoV) by Real-Time RT-PCR.” Eurosurveillance 25, no. 3 (January 23, 2020): 2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045a.

ARTIC v3: Tyson, John R., Phillip James, David Stoddart, Natalie Sparks, Arthur Wickenhagen, Grant Hall, Ji Hyun Choi, et al. “Improvements to the ARTIC Multiplex PCR Method for SARS-CoV-2 Genome Sequencing Using Nanopore.” BioRxiv: The Preprint Server for Biology, September 4, 2020. https://doi.org/10.1101/2020.09.04.283077.

Midnight 1200 bp: Freed, Nikki E., Markéta Vlková, Muhammad B. Faisal, and Olin K. Silander. “Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 Using 1200 Bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding.” Biology Methods & Protocols 5, no. 1 (2020): bpaa014. https://doi.org/10.1093/biomethods/bpaa014.

ARTIC v4, v4.1: ARTIC Real-time Genomic Surveillance. “SARS-CoV-2 Version 4 Scheme Release - Laboratory,” June 24, 2021. , https://community.artic.network/t/sars-cov-2-version-4-scheme-release/312 https://community.artic.network/t/sars-cov-2-v4-1-update-for-omicron-variant/342

VarSkip: VarSkip Multiplex PCR Designs for SARS-CoV-2 Sequencing. 2021. Reprint, New England Biolabs Inc., 2021. https://github.com/nebiolabs/VarSkip".

Resende: Resende, Paola Cristina, Fernando Couto Motta, Sunando Roy, Luciana Appolinario, Allison Fabri, Joilson Xavier, Kathryn Harris, et al. “SARS-CoV-2 Genomes Recovered by Long Amplicon Tiling Multiplex Approach Using Nanopore Sequencing and Applicable to Other Sequencing Platforms,” May 1, 2020. https://doi.org/10.1101/2020.04.30.069039.

USydney: Eden, John-Sebastian, and Eby Sim. “SARS-CoV-2 Genome Sequencing Using Long Pooled Amplicons on Illumina Platforms.” protocols.io, April 4, 2020. https://www.protocols.io/view/sars-cov-2-genome-sequencing-using-long-pooled-amp-befyjbpw.

Swift Swift BioSciences. “NEW- Swift Normalase™ Amplicon SARS-CoV-2 Panels.” Accessed December 2, 2021. https://swiftbiosci.com/swift-normalase-amplicon-sars-cov-2-panels-3/".

UNZA Simulundu, Edgar, Francis Mupeta, Pascalina Chanda-Kapata, Ngonda Saasa, Katendi Changula, Walter Muleya, Simbarashe Chitanga, et al. “First COVID-19 Case in Zambia — Comparative Phylogenomic Analyses of SARS-CoV-2 Detected in African Countries.” International Journal of Infectious Diseases 102 (January 1, 2021): 455–59. https://doi.org/10.1016/j.ijid.2020.09.1480.

Software Tools:

Li, Heng. “Minimap2: Pairwise Alignment for Nucleotide Sequences.” Bioinformatics 34, no. 18 (September 15, 2018): 3094–3100. https://doi.org/10.1093/bioinformatics/bty191 .

Pangolin. Python. 2020. Reprint, CoV-lineages, 2022. https://github.com/cov-lineages/pangolin.

Gofasta. Go. 2020. Reprint, virus-evolution, 2022. https://github.com/virus-evolution/gofasta.

Köster, Johannes, and Sven Rahmann. “Snakemake—a Scalable Bioinformatics Workflow Engine.” Bioinformatics 28, no. 19 (October 1, 2012): 2520–22. https://doi.org/10.1093/bioinformatics/bts480 .

Scorpio. Python. 2021. Reprint, CoV-lineages, 2022. https://github.com/cov-lineages/scorpio.

Garrison, Erik, and Gabor Marth. “Haplotype-Based Variant Detection from Short-Read Sequencing.” ArXiv:1207.3907 [q-Bio], July 20, 2012. http://arxiv.org/abs/1207.3907.

Quinlan, Aaron R, and Ira M Hall. “BEDTools: A Flexible Suite of Utilities for Comparing Genomic Features.” Bioinformatics (Oxford, England) 26 (2010): 841–42. https://doi.org/10.1093/bioinformatics/btq033 .

Geneious version 2022.0.2 created by Biomatters. Available from https://www.geneious.com.

Robinson, James T, Helga Thorvaldsdottir, Douglass Turner, and Jill P Mesirov. “igv.js: An Embeddable JavaScript Implementation of the Integrative Genomics Viewer (IGV).” Bioinformatics 39, no. 1 (January 1, 2023): btac830. https://doi.org/10.1093/bioinformatics/btac830.

History:

2022-02-11: added VarSkip v2, condensed Delta lineages into a single composite track, added BA.1.1 and BA.2

2021-12-10: added additional requested primer sets (ARTICv4.1, USydney, Resende, Swift, UNZA), updated omicron nomenclature

2021-11-29: added missing diagnostic tests

2021-11-27: added currently circulating lineages, new primer overlap visualization

2021-10-07: removed duplicate VarSkip amplicon 61

2021-09-29: Added VarSkip, removed new VBM lineages

2021-06-24: Added ARTICv4

2021-06-20: Updated to currently relevant variants of concerns.