key: cord-0773223-6tqa6xqx
authors: Hickman, R.; Nguyen, J.; Lee, T. D.; Tyson, J. R.; Azana, R.; Tsang, F.; Hoang, L.; Prystajecky, N.
title: Rapid, High-Throughput, Cost Effective Whole Genome Sequencing of SARS-CoV-2 Using a Condensed One Hour Library Preparation of the Illumina DNA Prep Kit
date: 2022-02-08
journal: nan
DOI: 10.1101/2022.02.07.22269672
sha: f9789e95c77d4ca780cc82fed72951ad1f493454
doc_id: 773223
cord_uid: 6tqa6xqx

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely genomic sequencing of SARS-CoV-2 viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. Additionally, the enormous demand of genomic sequencing on supply chains and the resulting shortages of laboratory supplies necessitate the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method where the same protocol can be used in a STAT scenario, from sample to sequencer in as little as eight hours, and a high-throughput scenario, where one technologist can perform 576 library preparations over the course of one 8-hour shift. This new method uses the Freed et al. 1200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and truncated Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original, application of this new method in hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results suggest that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 whole genome sequencing protocol.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for the 41 novel 2019 coronavirus disease (COVID-19), is responsible for the pandemic first identified in 42 Wuhan, China, and since has spread worldwide. As of June 2nd 2021, there have been over 171 43 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22269672 doi: medRxiv preprint 3 million cases and 3.5 million COVID-19 deaths confirmed (https://coronavirus.jhu.edu/), and the 44 pandemic continues to have devastating social, health, and economic impacts globally. The 45 reliance on genomics for the study of viral transmission, identifying variants of interest (VoI) 46 and of concern (VoC), and for studying vaccine breakthrough has necessitated rapid, high-47 throughput, and high-quality whole genome sequencing. This has resulted in unprecedented 48 demand for laboratory reagents, consumables, equipment, and highly skilled laboratory staff to 49 generate sequence data. With global shortages of laboratory reagents and consumables, there is 50 widespread need for methods with low-reagent and low-consumable requirements that are 51 scalable.

A multiplexed 1200 bp tiled primer amplicon approach previously described (1) , which 53 generates high and consistent coverage amplification across the SARS-CoV-2 genome, was 54 chosen for best compatibility with the DNA Prep library preparation kit (Illumina, CA, USA) 55 compared to other widely used primer sets (2, 3). Illumina's DNA Prep library preparation kit 56 has been shown to generate high-quality, robust library preparations with a method that is 57 scalable and liquid handler compatible. However, this library preparation method is 58 comparatively expensive and more complex than alternatives. Here we use an optimized and 59 truncated Illumina DNA Prep method that minimizes cost, hands-on time, and complexity of 60 work while maintaining high-quality and robust sequence data. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The optimized library preparation method, performed at half reaction volumes, eliminates 81 amplicon purification, tagmentation stop, and post-tagmentation washes entirely ( Fig 1B) . Briefly, the multiplex primer pools were combined. Next, 15µl of amplicon, 5µl of bead linked 86 transposomes (BLT) and 5µl of tagmentation buffer 1 (TB1) were heated on a thermal cycler at 87 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22269672 doi: medRxiv preprint 55⁰C for 15 minutes. On a magnet, the supernatant was removed and taken off the magnet, the 88 beads were then re-suspended in 10µl of enhanced PCR mix (EPM), 10µl of ultrapure water, and 89 5µl of Illumina's Unique Dual Indexes before being run on a reduced 8-cycle library PCR. After Reports describing sequencing quality metrics-including genome completeness, depth of 109 coverage, and quality flags-and lineage information were generated using ncov-tools from the 110 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) (Fig 2) .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Eppendorf epMotion 5075t instrument (1.5 hours vs. 6.5 hours, respectively) (Table 2, Fig 1B) .

The new method was also associated with a lower cost of library preparation ($31 vs. $68) and 143 required fewer tip boxes (4 vs. 34) compared to the original method (Table 2, Fig 1B) . 

In conclusion, this study demonstrates a rapid library preparation method for SARS-CoV-2 187 whole genome sequencing that produces consistent and high-quality data with equivalent results 188 to the original full length DNA Prep library preparation as described from the manufacturer.

With an overall savings of >75% hands-on time at 45% the cost and using 15% of the 

The authors declare no conflicts of interest.

This study did not receive any funding.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22269672 doi: medRxiv preprint

Rapid and inexpensive whole-222 genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford 223

nCoV-2019 sequencing protocol v3 (LoCost)

Improvements to the ARTIC multiplex PCR method for 229 SARS-CoV-2 genome sequencing using nanopore

Nextera DNA Flex Library Preparation Reference Guide, document 231 1000000025416 v07. Illumina

Pybus 236 OG. 2020. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist 237 genomic epidemiology

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted February 8, 2022. ; https://doi.org/10.1101/2022.02.07.22269672 doi: medRxiv preprint