A year of sequencing COVID-19

 
 

In Spring 2021, ʻĀina-Informatics partnered with Dr. Ed Desmond (Hawaiʻi Department of Health) and Dr. Marguerite Butler (University of Hawaiʻi at Mānoa, Pacific Biodiversity Lab) to design the COVID Trackers Project, a lab-based curriculum which enabled students to use MinION sequencing technology to contribute towards variant assignment of SARS-CoV-2 samples. All test samples were isolated from individuals who tested positive at State testing locations between 4/22/2021 and 8/31/2021 (capturing the Delta surge in Hawaiʻi) and only brought into classrooms once processed and deactivated by our DOH and UHM collaborators.

Dr. Marguerite Butler (University of Hawaiʻi at Mānoa - College of Natural Sciences)

Dr. Ed Desmond (Hawaiʻi Department of Health - Public Health and Environmental Laboratories)

The in-classroom activities were enabled by the use of a custom mobile sequencing lab outfitted with all the necessary equipment and reagents to conduct this outreach. Lab curricula were designed around the ARTIC Network workflow for processing and sequencing SARS-CoV-2 using a tiled amplicon approach. Beginning with amplicons generated by the Butler Lab, students first constructed multiplex sequencing libraries pooling 6 genomes at a time with NEB’s ARTIC SARS-CoV-2 Companion Kit for Oxford Nanopore Technologies. During the course of this project, our collaborators in the Butler Lab utilized multiple primer schemes (including VarSkip and VarSkip v2) for amplicon generation as each scheme became available. The majority of the sequencing runs were performed on MinION Flongle flow cells, with a smaller subset completed using standard MinION flow cells. Reads were analyzed in real time in the classroom using RAMPART, with final assembly and variant confirmation conducted in Medaka and Pangolin outside of the classroom. A portion of these consensus sequences were contributed to GISAID in Spring 2022. Finally, students ran a biogeographical analysis of 212 publicly available SARS-CoV-2 genomes relevant to Hawaiʻi using data and R scripts developed by Ethan Hill (Butler Lab) and powered by RStudio Cloud.

Ethan Hill (Butler Lab, UH Mānoa) coaching a ʻIolani School student in COVID sequencing library preparation.

Photo credits: Eric Wehner

Global Health teacher Nan Ketpura-Ching working with students on a COVID-19 genome assembly puzzle.

An excerpt from a biogeographic reconstruction generated by students in RStudio Cloud depicting chains of transmission of COVID into and through our islands.


This project has been generously supported in part by a Governor’s Emergency Educational Relief Grant as well as a sponsorship from Hawaiʻi Dental Service.

Schools and student reach

The COVID Trackers Lab was conducted at 14 participating schools on three islands during SY2021-22, reaching over 640 local students. Of these, 2 were public middle schools, 8 were public high schools, 2 were public charter schools and 2 were private schools.

 
School Island Genomes
Attempted		 Participating
Teachers		 Student
Reach
ʻIolani School Oʻahu 114 2 24
ʻAiea High School Oʻahu 12 1 17
Waipahu High School Oʻahu 12 1 28
King Intermediate School Oʻahu 24 2 83
Kailua Intermediate School Oʻahu 30 1 90
Kahuku High School Oʻahu 60 2 208
Hawaiʻi Baptist Academy Oʻahu 96 2 15
Kailua High School Oʻahu 12 1 22
Hawaiʻi Technology Academy Kona Hawaiʻi 6 1 8
Kapaʻa High School Kauaʻi 18 2 28
Hawaiʻi Technology Academy Kauaʻi Kauaʻi 6 1 19
Kauaʻi High School Kauaʻi 6 1 20
Radford High School Oʻahu 12 1 46
Hilo High School Hawaiʻi 12 1 35
TOTALS 420 19 643
 

A student at King Intermediate School preparing a sequencing library for loading on a MinION Flongle flow cell.

Photo credit: E. Tong

Data

The student generated data include a total of 236 unique samples sequenced in classrooms 6 or 12 at a time plus two additional 96-well plates. A subset of the 236 unique samples (n = 84) were resequenced in classrooms when prior sequencing runs in the Butler Lab yielded insufficient reads, allowing for multiple datasets to be merged for a more robust combined sequencing depth.

A summary of student-generated variant assignments by sampling week (96 well plate samples omitted).

The vast majority of samples sequenced were taken from surge testing efforts on or around 8/12/2021, with results capturing the Delta surge across the islands. Approximately 70% of all samples originated on Oʻahu, 20% in Hawaiʻi County, and 5% each in Kauaʻi and Maui counties.

Reflections

The disruption to school and student learning brought upon by the COVID pandemic may not yet be fully understood, but the pandemic forced all of us to think outside the box and innovate new ways to engage students both in and out of the classroom. At the same time, the DOH was facing a shortage of skilled technicians crucial in developing the sequencing infrastructure required to conduct genomic surveillance of a new virus. This unique confluence of problems was met head on with a bold idea: to train the next generation of genome scientists in the midst of an ongoing genome science crisis.

With huge mahalo for our academic and funding partners, the ʻĀina-Informatics Network rallied around this bold idea, committing countless hours and resources in order to empower students to join the pandemic response. In a year where COVID held control over their everyday lives, students found a way to assert agency over COVID using a pipette, a sequencer and a laptop.

Kailua High School students presenting a completed COVID genome assembly puzzle designed to illustrate the evolution of new variants.

Photo credit: Sara Anglin

The pandemic also opened up new case studies in bioethics, especially surrounding the novel mRNA vaccines developed with unthinkable speed in response to variant after variant. Ripped straight from the headlines, our bioethics unit engaged students in considering the ethics surrounding the initially limited vaccine rollout, subsequent mandating of vaccines in the workplace and the inequities in the global distribution of vaccine access.

We are grateful for the opportunity to have brought this project to so many in our community of schools, but we are also relieved that the pandemic is at a place where genomic surveillance at this scale is no longer necessary. And while the pandemic brought with it heartache and suffering, it has also presented a unique opportunity for our program to directly engage students in genome science in the most timely and urgent way possible. It is our hope that among the students we reached with this project is a new generation of homegrown genome science professionals ready to lead us through the next crisis when it arrives.

Genomic sequencing by ʻAiea High School students identifies multiple variants in Hawaiʻi swabs

by Sarah Gallardo, ʻAiea High School Medical Biotechnology student

Teacher: Mrs. Mary Margaret Peterson

What did you do on the four days of the project?

For four days we worked with instructors from ‘Iolani and UH to identify the type of variant of twelve different COVID-19 samples which originated throughout the state of Hawai’i around late June to early July of 2021. Using the given genetic material, we created a mixture to produce a single nucleotide overhang where the barcodes, which identifies each sample, can be attached. In the next step we attached distinct barcodes assigned to each of the twelve samples.

Students preparing a sequence library for barcoding.

Photo credit: Mary Margaret Peterson

Then we combined all of the barcoded libraries into a single mixed library. Before putting the samples through a DNA sequencer, we had to get the DNA in its purest form which we did with a process called DNA purification. We then sequenced the library with nanopore sequencing. This is where “a nanopore, a synthetic membrane channel, allows a strand of DNA through its pore. As the molecule passes, changes in the electrical field are detected.” This is decoded resulting in a DNA sequence. 

Why is it important to track variants in Hawaiʻi and around the world? 

We can identify which variants are more transmissible than others thus becoming more proactive when controlling outbreaks. We can understand how the coronavirus arrives in our community and how it moves around within the state. The data retrieved from genomic surveillance helps track mutations occurring within the SARS-CoV-2 genome as it transfers from infected people. 

What variants did your class find?

In viewing our results, we found

one new case of Gamma,

three cases of Alpha,

seven samples of Delta,

and one of Mu.

This proves that during late June and early July of 2021 the Delta variant started to become the most transmissible and common variant in the population. 

Student preparing sample for loading on the MinION sequencer.

Photo credit: E. Tong

Write about how you felt completing the project. What did you learn?

This experience involved an immense amount of complex thinking and being exposed to new concepts and scientific terminology. I’m grateful that I had this opportunity to partake in a process that most people my age don’t get to experience until college. Through the guidance of Mr. Tong and Mr. Hill, I became more aware of how the coronavirus is traveling within the state of Hawai’i and the importance of genomic surveillance. Knowing that my class and I were contributing data to our current health crisis is extremely rewarding.

Chasing the Variants

by Katelyn Shu, ʻIolani School Global Health student

Teacher: Mrs. Nan Ketpura-Ching

This past week my classmates and I have been fortunate enough to participate in a COVID-19 Variant Trackers Project with ʻĀina-Informatics. Viruses like SARS-CoV-2 are constantly mutating and changing into new variants. When the highly contagious delta variant came to Hawaiʻi, the resulting surge during the summer had COVID case numbers higher than ever before. Part of the job of someone who specializes in bioinformatics is to use computer tools to analyze biological data, which can be used to learn about these variants and how they spread around the world.

We began the project learning a little bit about how SARS-CoV-2 is sequenced to find the order of the nucleotide bases that make up the RNA. Many runs are taken to be more accurate. If when it’s being sequenced, a base doesn’t match up with the reference genome, it could be a possible error or mutation. The method of sequencing we were able to see with ʻĀina-Informatics was called Nanopore sequencing, where DNA goes through nanopores that detect electrical changes with sensor arrays. These signals can be read by a flowcell device. We were provided with different samples of swabs from people who contracted COVID-19 during June-July 2021 on Oʻahu. Some of my classmates got to use micropipettes to put the samples into the flowcells.

Taking a closer look at a MinION flow cell.

Photo credits: Eric Wehner

Priming the flow cell for sequencing.

When we returned later, we were able to learn about the programming tool called RStudio. Biologists use RStudio to do complex statistical analysis. Using this tool, Mr. Tong helped us create a “family tree” of sorts for the spread of COVID-19. It’s really fascinating. By sequencing countless nucleotides and tracking the mutations, biologists can track how variants travel based on the similarities between the COVID genomes of swabbed people. I was so fascinated to look at the family tree that was generated. In the end, our references showed that most of the samples we found were the Delta variant. A few others were Alpha or Gamma, but there were no samples from the original Wuhan strain nor the Mu and Beta variants. This fit our hypothesis that most of the samples would be Delta.

Variant assignment of genomic reads in RAMPART.

Photo credit: Eric Wehner

On top of the contact-tracing we learned about during the epidemiology unit, these programs can truly reveal a whole new side to the broad spectrum that is biology. While others interview people and try to piece together correlations between infected people, people in bioinformatics have extremely important jobs as well in biologically mapping out the path of the pathogen.

UH Mānoa researchers share how genomic data are used in pandemic response

At our September 15 STEMplus virtual launch, UH evolutionary biologists Ethan Hill and Dr. Marguerite Butler (Pacific Biodiversity Lab) presented on how we can detect which SARS-CoV-2 variants are circulating around Hawaiʻi. First, Ethan shares how students can safely contribute towards genomic surveillance of the virus. From the mutations observed between samples, students can then use bioinformatics tools to reconstruct the many chains of transmission across the islands. Dr. Butler then updates us on the state of variants in Hawaiʻi, and breaks down how variant tracking is used in our pandemic response.

ʻĀina-Informatics discusses the new COVID Variant Trackers Project on HPR's Bytemarks Cafe

Mahalo nui to Bytemarks Cafe host Burt Lum for inviting ʻIolani teachers Eric and Joanna on to the radio to talk about the new COVID Variant Trackers Project from ʻĀina-Informatics. Learn how our mobile lab can bring SARS-CoV-2 genome sequencing safely into the hands of students across the pae ʻāina. Our segment begins at the 7:14 time mark.

 

Photo credit: Burt Lum