Methodology and Protocol
Biobot’s Covid-19 wastewater sequencing product
For information on Biobot’s Covid-19 wastewater qPCR product, please visit biobot.io/covid19-report-notes
Extraction protocol
Our methods for extracting SARS-CoV-2 from wastewater rely on state-of-the-art protocols. Briefly, we perform virus capture using magnetic nanoparticles (Ceres NanoTrap), and then conduct RNA extraction. The extracted viral RNA is then used for downstream measurements (both qPCR and sequencing).
Data use
Biobot’s wastewater data provides a complementary metric to guide response to the Covid-19
outbreak. We recommend sharing this information with local public health officials. We believe this work has the greatest impact on a statewide level, and hope that you will reach out to your local officials and encourage the expansion of our partnership across your state.
For questions specific to your report, email support@biobot.io
Sequencing Protocol
To prepare for sequencing, we generate complementary DNA from extracted RNA. DNA is then provided to our trusted partners for library preparation using the ARTIC V5.3 primer set. Sequencing is conducted using the Illumina NextSeq instrument (paired end 2×101 reads).
Sequencing Analysis
We analyze sequence data to determine the abundances of SARS-CoV-2 variants present and to ensure strict quality criteria are met. Bioinformatics include filtering to remove substandard reads, alignment of reads to the SARS-CoV-2 genome, and estimation of variant abundances using the Freyja software.
Release notes
Extraction protocol versions
We are continuously working to improve our protocols to increase the sensitivity of our measurements and reduce variability. You can find the protocol that was used to generate your data at the bottom of each page of your report:
The extraction protocol version is tracked here, under “lab protocol versions.”
Sequencing protocol versions
To accommodate changes in the SARS-CoV-2 genome, we use up-to-date, proven primer sets to prepare for sequencing. Our trusted sequencing partners use Illumina instruments.
| SEQUENCING PROTOCOL VERSION | DATE | SEQUENCING PARTNER | INSTRUMENT | PRIMER SET | DESCRIPTION |
| v1.4 (current) | 5/15/2023 | Ginkgo Bioworks | Illumina NovaSeq 6000 (2 x 101) | ARTIC v.5.3 | Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner |
| v1.3 | 4/25/2022 | Ginkgo Bioworks | Illumina NovaSeq 6000 (2 x 101) | ARTIC V4.1 | Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner. |
| v1.2 | 12/30/2021 | Ginkgo Bioworks | Illumina NovaSeq 6000 (2 x 101) | ARTIC V4 | Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner. |
| v1.1 | 07/02/2021 | Ginkgo Bioworks | Illumina NovaSeq 6000 (2 x 101) | ARTIC V3 | Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner. |
Sequencing analysis versions
Bioinformatics include ensuring data meet stringent quality criteria and estimation of the relative abundances of SARS-CoV-2 variants within a particular sample. We keep a close eye on the latest bioinformatic developments to ensure our pipeline is state-of-the-art.
| SEQUENCING ANALYSIS VERSION | DATE | DESCRIPTION |
| v2.0 (current) | 06/03/2022 | Bioinformatics include read filtering to meet stringent quality criteria, alignment to the SARS-CoV-2 genome, and variant relative abundance determination. With v2.0 we expanded quality control measures and implemented a more flexible approach, Freyja for variant quantification. |
| v1.0 | 12/12/2021 | Bioinformatics include read filtering to meet stringent quality criteria, alignment to the SARS-CoV-2 genome, and variant relative abundance determination. |
Reported variants – Weekly Variant Percentages Report
| SEQUENCING ANALYSIS VERSION | DATE | VARIANTS OF CONCERN REPORTED | DESCRIPTION |
| v1.14 (current) | 04/08/2024 | (Omicron) • XBB • XBB.1.5 • XBB.1.9 • EG.5 • BA.2.86 • JN.1 • JN.1.13 Other variants | Added: • JN.1.13 Removed: • HV.1 • FL.1.5.1 All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes sublineages XBB.1.42, etc. “Other variants” contains all variants not explicitly quantified. |
| v1.13 | 12/20/2023 | (Omicron) • XBB • XBB.1.5 • XBB.1.9 • FL.1.5.1 • EG.5 • HV.1 • BA.2.86 • JN.1 Other variants | Added: • JN.1 • BA.2.86 Removed: • XBB.1.9.1 • XBB.1.9.2 • XBB.1.16 All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes sublineages XBB.1.42, etc. “Other variants” contains all variants not explicitly quantified. |
| v1.12 (current) | 12/04/2023 | (Omicron) • XBB • XBB.1.5 • XBB.1.9.1 • FL.1.5.1 • XBB.1.9.2 • EG.5 • HV.1 • XBB.1.16 Other variants | Added: • HV.1 Removed: • XBB.1.9 All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes sublineages XBB.1.9.2, XBB.1.42, etc. “Other variants” contains all variants not explicitly quantified. |
| v1.11 | 09/28/2023 | (Omicron) • XBB* • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • XBB.1.16 • EG.5 • FL.1.5.1 Other variants | Added: • FL.1.5.1 XBB* includes all XBB sublineages aside from those specifically reported. “Other variants” contains all variants not explicitly quantified. |
| v1.10 | 07/20/2023 | (Omicron) • XBB* • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • XBB.1.16 • EG.5 Other variants | Added: • XBB.1.5 • XBB.1.9.1 • XBB.1.9.2 • EG.5 XBB* includes all XBB sublineages aside from those specifically reported. “Other variants” contains all variants not explicitly quantified. Removed: • BA.2* • BA.5* • BQ.1* • XBB* |
| v1.9 | 04/28/2023 | (Omicron) • BA.2* • BA.5* • BQ.1* • XBB* • XBB.1.9 • XBB.1.16 Other variants | Split XBB* into XBB*, XBB.1.16 and XBB.1.9. XBB* includes all XBB lineages aside from 1.9 and 1.16 Removed: BF.7 BA.4* |
| v1.8 | 01/10/2023 | (Omicron) • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* Other variants | BA.2* includes BA.2, BA.2.12, BA.2.12.1, and other BA.2 sub-lineages. BA.4* includes BA.4 and its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, including XBB.1.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. “Other variants” contains all variants not explicitly quantified. |
| v1.7 | 12/14/2022 | (Omicron) • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB Other variants | BA.2* includes BA.2, BA.2.12, BA.2.12.1, and other BA.2 sub-lineages. BA.4* includes BA.4 and its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. “Other variants” contains all variants not explicitly quantified. |
| v1.6 | 11/01/2022 | (Omicron) • BA.2* • BA.4* • BA.5 • BF.7 • BQ.1* Other variants | BA.2* includes BA.2, BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and BA.4.6. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. |
| v1.5 | 10/27/2022 | (Omicron) • BA.2* • BA.4* • BA.5 • BF.7 Other variants | BA.2* includes BA.2, BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and BA.4.6. A notation change to emphasize that a single asterisk indicates that sublineages are included in a variant, which are specified in release notes. |
| v1.4 | 10/06/2022 | (Omicron) • BA.2* • BA.4** • BA.5 Other variants | *Includes BA.2, BA.2.12 and BA.2.12.1 **Includes BA.4 and BA.4.6. Notation change to emphasize that sublineages of BA.4, including BA.4.6, are included in abundance estimates for the BA.4 variant. |
| v1.3 | 07/20/2022 | (Omicron) • BA.2* • BA.4 • BA.5 Other variants | Removed: *Individual reporting on BA.2, BA.2.12 and BA.2.12.1 (now combined) |
| v1.2 | 07/08/2022 | (Omicron) • BA.2 subvariants ㅤ➞ Other BA.2 ㅤ➞ BA.2.12 • BA.4 • BA.5 Other variants | Added: Split out BA.4 from BA.5 Removed: BA.1 |
| v1.1 | 06/03/2022 | (Omicron) • BA.1 • BA.2 subvariants ㅤ➞ Other BA.2 ㅤ➞ BA.2.12 • BA.4/5 Other variants | Added: BA.2.12, including BA.12.1 sublineages, and a combined percentage of BA.4 and BA.5 as variants of concern Removed: Delta as it wasn’t a significant contributor |
| Delta Omicron • BA.1/1.1 • BA.2 | First variant report delivered results on variants of concern, especially Omicron subvariant thought to be more transmissible (BA.2) |
Reported variants – Biobot Network Visualization (biobot.io/data)
| SEQUENCING ANALYSIS VERSION | DATE | VARIANTS OF CONCERN REPORTED | DESCRIPTION |
| v1.18 (current) | 04/08/2024 | Regional overview: • Delta • BA.1 • BA.2 • BA.2.12 • BA.4 • BA.5 • BF.7 • BQ.1 • XBB • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • HV.1 • FL.1.5.1 • JN.1 • JN.1.13 • Other variants | All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes XBB.1.42, BQ.1 contains BQ.1.1. etc. Special note on XBB: The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: JN.1.13 at the regional level |
| v1.17 | 12/04/2023 | Regional overview: • Delta • BA.1 • BA.2 • BA.2.12 • BA.4 • BA.5 • BF.7 • BQ.1 • XBB • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • HV.1 • FL.1.5.1 • JN.1 • Other variants | All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes XBB.1.42, BQ.1 contains BQ.1.1. etc. Special note on XBB: The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: JN.1 at the regional level |
| v1.16 | 12/04/2023 | Regional overview: • Delta • BA.1 • BA.2 • BA.2.12 • BA.4 • BA.5 • BF.7 • BQ.1 • XBB • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • HV.1 • FL.1.5.1 • Other variants | All variants contain sublineages (aside from those specifically reported separately). E.g. XBB includes XBB.1.42, BA.2 contains BA.2.86, BQ.1 contains BQ.1.1. etc. Special note on XBB: The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: HV.1 at the regional level |
| v1.15 | 09/28/2023 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • FL.1.5.1 • Other variants | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1. BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, except 1.5, 1.9, 1.9.1, 1.9.2, 1.16, and EG.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: FL.1.5.1 at regional-level |
| v1.14 | 08/18/2023 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • Other variants | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1. BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, except 1.5, 1.9, 1.9.1, 1.9.2, 1.16, and EG.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Removed: XBB, XBB.1.9, and XBB.1.16 at county-level |
| v1.13 | 07/20/2023 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* • XBB.1.16 • XBB.1.5 • XBB.1.9 • XBB.1.9.1 • XBB.1.9.2 • EG.5 • Other variants County: • XBB* • XBB.1.9 • XBB.1.16 | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1. BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, except 1.5, 1.9, 1.9.1, 1.9.2, 1.16, and EG.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: XBB.1.5, XBB.1.9.1, XBB.1.9.2, and EG.5 at regional-level |
| v1.12 | 04/28/2023 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* • XBB.1.16 • XBB.1.9 • Other variants County: • XBB* • XBB.1.9 • XBB.1.16 | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1. BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, except 1.9 and 1.16, including XBB.1.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: XBB.1.9 and XBB.1.16 at regional and county levels Removed: BQ.1* and BA.5* from the county level |
| v1.11 | 01/10/2023 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB* • Other variants County: • BQ.1* • XBB* • BA.5* | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. XBB* includes all XBB sublineages, including XBB.1.5. The progenitor lineages of XBB are BA.2.75 and BA.2.10. Since neither progenitor is circulating, we combine abundances of genomic fragments of these BA.2 lineages with XBB. Added: XBB* at regional level XBB* at county level Removed: BA.4 from the county level |
| v1.10 | 12/14/2022 | Regional overview: • BA.2* • BA.4* • BA.5* • BF.7 • BQ.1* • XBB • Other variants County: • BQ.1* • BA.4* • BA.5* | BA.2* includes BA.2 and all its sublineages, including BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and all its sublineages, including BA.4.6. BA.5* includes the sublineages of BA.5, except BQ.1* and BF.7. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. |
| v1.9 | 11/01/2022 | Regional overview: • BA.2* • BA.4* • BA.5 • BF.7 • BQ.1* • Other variants County: • BQ.1* • BA.4* • BA.5 | BA.2* includes BA.2, BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and BA.4.6. BQ.1* includes BQ.1 and its sublineages, including BQ.1.1. |
| v1.8 | 10/27/2022 | Regional overview: • BA.2* • BA.4* • BA.5 • BF.7 • Other variants County: • BA.2* • BA.4* • BA.5 | BA.2* includes BA.2, BA.2.12 and BA.2.12.1 BA.4* includes BA.4 and BA.4.6. A notation change to emphasize that a single asterisk indicates that sublineages are included in a variant, which are specified in release notes. |
| v1.7 | 10/13/2022 | Regional overview: • BA.2* • BA.4** • BA.5 • Other variants County: • BA.2* • BA.4** • BA.5 | *Includes BA.2, BA.2.12 and BA.2.12.1 **Includes BA.4 and BA.4.6. Notation change to emphasize that sublineages of BA.4, including BA.4.6, are included in abundance estimates for the BA.4 variant. |
| v1.6 | 07/21/2022 | Regional overview: • BA.2* • BA.4 • BA.5 • Other variants County: • BA.2* • BA.4 • BA.5 | Removed: *Individual reporting on BA.2, BA.2.12 and BA.2.12.1 (now combined) *Condensed label for BA.2/BA.2.12 at the county level. |
| v1.5 | 07/08/2022 | Regional overview: • BA.2 • BA.2.12 • BA.4 • BA.5 • Other County: • BA.2/BA.2.12 • BA.4 • BA.5 | Added: • Split out BA.4 from BA.5 for the regional-level and county levels Removed: • BA.1 from regional-level • Collapsed BA.2/BA.2.12 at the county-level |
| v1.4 | 06/15/2022 | Regional overview: • BA.1 • BA.2 • BA.2.12 • BA.4/5 • Other County: • BA.2 • BA.2.12 • BA.4/5 | Added: BA.4/5 to county-level Removed: BA.1 from county-level |
| v1.3 | 06/03/2022 | Regional overview: • BA.1 • BA.2 • BA.2.12 • BA.4/5 • Other County: • BA.1 • BA.2 • BA.2.12 | Added: • BA.2.12, including BA.12.1 sublineages to the regional and county levels • Combined percentage of BA.4 and BA.5 to the regional overview as variants of concern Removed: • Delta from both the regional and county levels as it wasn’t a significant contributor |
| v1.2 | 02/24/2022 | Regional overview: • Delta • Omicron ㅤ➞ BA.1/1.1 ㅤㅤ➞ BA.2 County: • Delta • BA.1/1.1 • BA.2 | Added: Subvariants of Omicron on the regional and county levels (BA.1 & BA1.1) and BA.2 |
| v1.1 | 02/17/2022 | Regional overview: • Delta • Omicron County: • Delta • Omicron | Added: Variant percentages of Omicron and Delta to county-level graphs |
| v1.0 | 02/03/2022 | Regional overview: • Delta • Omicron | First variant reporting on biobot.io/data delivered results on two variants of concern at the regional level: Omicron and Delta |