Efficient high-throughput SARS-CoV-2 testing to detect asymptomatic carriers

Recent reports suggest that 10 to 30% of severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) infected patients are asymptomatic and that viral shedding may occur before symptom onset. Therefore, there is an urgent need to increase diagnostic testing capabilities to prevent disease spread. We developed P-BEST, a method for Pooling-Based Efficient SARS-CoV-2 Testing, which identifies all positive subjects within a set of samples using a single round of testing. Each sample is assigned into multiple pools using a combinatorial pooling strategy based on compressed sensing. We pooled sets of 384 samples into 48 pools, providing both an eightfold increase in testing efficiency and an eightfold reduction in test costs, while identifying up to five positive carriers. We then used P-BEST to screen 1115 health care workers using 144 tests. P- BEST provides an efficient and easy-to-implement solution for increasing testing capacity that can be easily integrated into diagnostic laboratories.

Tapestry: A Single-Round Smart Pooling Technique for COVID-19 Testing

The COVID-19 pandemic has strained testing capabilities worldwide. There is an urgent need to find economical and scalable ways to test more people. We present Tapestry, a novel quantitative nonadaptive pooling scheme to test many samples using only a few tests. The underlying molecular diagnostic test is any real-time RT-PCR diagnostic panel approved for the detection of the SARS-CoV-2 virus. In cases where most samples are negative for the virus, Tapestry accurately identifies the status of each individual sample with a single round of testing in fewer tests than simple two-round pooling. We also present a companion Android application BYOM Smart Testing which guides users through the pipetting steps required to perform the combinatorial pooling. The results of the pooled tests can be fed into the application to recover the status and estimated viral load for each individual sample.NOTEThis protocol has been validated with in vitro experiments that used synthetic RNA and DNA fragments and additionally, its expected behavior has been confirmed using computer simulations. Validation with clinical samples is ongoing. We are looking for clinical collaborators with access to patient samples. Please contact the corresponding author if you wish to validate this protocol on clinical samples.

Efficient high throughput SARS-CoV-2 testing to detect asymptomatic carriers

The COVID-19 pandemic is rapidly spreading throughout the world. Recent reports suggest that 10-30% of SARS-CoV-2 infected patients are asymptomatic. Other studies report that some subjects have significant viral shedding prior to symptom onset. Since both asymptomatic and pre-symptomatic subjects can spread the disease, identifying such individuals is critical for effective control of the SARS-CoV-2 pandemic. Therefore, there is an urgent need to increase diagnostic testing capabilities in order to also screen asymptomatic carriers. In fact, such tests will be routinely required until a vaccine is developed. Yet, a major bottleneck of managing the COVID-19 pandemic in many countries is diagnostic testing, due to limited laboratory capabilities as well as limited access to genome-extraction and Polymerase Chain Reaction (PCR) reagents. We developed P-BEST - a method for Pooling-Based Efficient SARS-CoV-2 Testing, using a non-adaptive group-testing approach, which significantly reduces the number of tests required to identify all positive subjects within a large set of samples. Instead of testing each sample separately, samples are pooled into groups and each pool is tested for SARS-CoV-2 using the standard clinically approved PCR-based diagnostic assay. Each sample is part of multiple pools, using a combinatorial pooling strategy based on compressed sensing designed for maximizing the ability to identify all positive individuals. We evaluated P-BEST using leftover samples that were previously clinically tested for COVID-19. In our current proof-of-concept study we pooled 384 patient samples into 48 pools providing an 8-fold increase in testing efficiency. Five sets of 384 samples, containing 1-5 positive carriers were screened and all positive carriers in each set were correctly identified. P-BEST provides an efficient and easy-to-implement solution for increasing testing capacity that will work with any clinically approved genome-extraction and PCR-based diagnostic methodologies.

Knockout Sudoku, a method for rapidly curating gene disruption collections

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of approximately $10,000. The method uses manual 4-dimensional combinatorial pooling, next-generation sequencing and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. Here we present a protocol for the generation, combinatorial pooling and annotation of highly oversampled progenitor collections and their subsequent algorithmically guided condensation and curation into high-quality collections suitable for rapid genetic screening and gene discovery.

Knockout Sudoku, a method for rapidly curating gene disruption collections

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of approximately $10,000. The method uses manual 4-dimensional combinatorial pooling, next-generation sequencing and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. Here we present a protocol for the generation, combinatorial pooling and annotation of highly oversampled progenitor collections and their subsequent algorithmically guided condensation and curation into high-quality collections suitable for rapid genetic screening and gene discovery.

Rapid Construction of a Whole-genome Transposon Insertion Collection for Shewanella oneidensis by Knockout Sudoku

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally they have needed an extraordinary technical effort to construct. Knockout Sudoku is a new method for directing the construction and purification of a curated whole-genome collection of singlegene disruption mutants generated by transposon mutagenesis. Using a simple combinatorial pooling scheme, a highly oversampled collection of transposon mutants can be condensed into a next-generation sequencing library in a single day. The identities of the mutants in the collection are then solved by a predictive algorithm based on Bayesian inference, allowing for rapid curation and validation. Starting from a progenitor collection of 39,918 transposon mutants, we compiled a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR–1 containing representatives for 3,667 genes. High-throughput kinetic measurements on this collection provide a comprehensive view of multiple extracellular electron transfer pathways operating in parallel.

Biological screens from linear codes: theory and tools

Molecular biology increasingly relies on large screens where enormous numbers of specimens are systematically assayed in the search for a particular, rare outcome. These screens include the systematic testing of small molecules for potential drugs and testing the association between genetic variation and a phenotype of interest. While these screens are ``hypothesis-free,'' they can be wasteful; pooling the specimens and then testing the pools is more efficient. We articulate in precise mathematical ways the type of structures useful in combinatorial pooling designs so as to eliminate waste, to provide light weight, flexible, and modular designs. We show that Reed-Solomon codes, and more generally linear codes, satisfy all of these mathematical properties. We further demonstrate the power of this technique with Reed-Solomon-based biological experiments. We provide general purpose tools for experimentalists to construct and carry out practical pooling designs with rigorous guarantees for large screens.

DECONVOLUTING BAC–GENE RELATIONSHIPS USING A PHYSICAL MAP

Deconvolution of relationships between bacterial artificial chromosome (BAC) clones and genes is a crucial step in the selective sequencing of regions of interest in a genome. It often includes combinatorial pooling of unique probes obtained from the genes (unigenes), and screening of the BAC library using the pools in a hybridization experiment. Since several probes can hybridize to the same BAC, in order for the deconvolution to be achievable the pooling design has to be able to handle a large number of positives. As a consequence, smaller pools need to be designed, which in turn increases the number of hybridization experiments, possibly making the entire protocol unfeasible. We propose a new algorithm that is capable of producing high-accuracy deconvolution even in the presence of a weak pooling design, i.e. when pools are rather large. The algorithm compensates for the decrease of information in the hybridization data by taking advantage of a physical map of the BAC clones. We show that the right combination of combinatorial pooling and our algorithm not only dramatically reduces the number of pools required, but also successfully deconvolutes the BAC–gene relationships with almost perfect accuracy. Software is available on request from the first author.