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 V4.1 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 VERSIONDATESEQUENCING PARTNERINSTRUMENTPRIMER SETDESCRIPTION
v1.3
(current)
5/15/2022Ginkgo BioworksIllumina NovaSeq 6000 (2 x 101)ARTIC V4.1Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner.
v1.2 12/30/2021Ginkgo BioworksIllumina NovaSeq 6000 (2 x 101)ARTIC V4Library preparation using the ARTIC primer set. Preparation performed by our sequencing partner.
v1.107/02/2021Ginkgo BioworksIllumina NovaSeq 6000 (2 x 101)ARTIC V3Library 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 VERSIONDATEDESCRIPTION
v2.0
(current)
06/03/2022Bioinformatics 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.
v1.012/12/2021Bioinformatics 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 VERSIONDATEVARIANTS OF CONCERN REPORTEDDESCRIPTION
v1.4
(current)
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.
v1.307/20/2022(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)
v1.207/08/2022(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
v1.106/03/2022(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 VERSIONDATEVARIANTS OF CONCERN REPORTEDDESCRIPTION
v1.6
(current)
07/21/2022Regional 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.
v1.507/08/2022Regional 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
v1.406/15/2022Regional 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
v1.306/03/2022Regional 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
v1.202/24/2022Regional 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
v1.102/17/2022Regional overview:
• Delta
• Omicron

County:
• Delta
• Omicron
Added:
Variant percentages of Omicron and Delta to county-level graphs
v1.002/03/2022Regional overview:
• Delta
• Omicron
First variant reporting on biobot.io/data delivered results on two variants of concern at the regional level: Omicron and Delta