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Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples

Bloom, Joshua S. and Sathe, Laila and Munugala, Chetan and Jones, Eric M. and Gasperini, Molly and Lubock, Nathan B. and Yarza, Fauna and Thompson, Erin M. and Kovary, Kyle M. and Park, Jimin and Marquette, Dawn and Kay, Stephania and Lucas, Mark and Love, TreQuan and Booeshaghi, A. Sina and Brandenberg, Oliver F. and Guo, Longhua and Boocock, James and Hochman, Myles and Simpkins, Scott W. and Lin, Isabella and LaPierre, Nathan and Hong, Duke and Zhang, Yi and Oland, Gabriel and Choe, Bianca Judy and Chandrasekaran, Sukantha and Hilt, Evann E. and Butte, Manish J. and Damoiseaux, Robert and Kravit, Clifford and Cooper, Aaron R. and Yin, Yi and Pachter, Lior and Garner, Omai B. and Flint, Jonathan and Eskin, Eleazar and Luo, Chongyuan and Kosuri, Sriram and Kruglyak, Leonid and Arboleda, Valerie A. (2021) Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples. Nature Biomedical Engineering, 5 (7). pp. 657-665. ISSN 2157-846X. PMCID PMC7480060. doi:10.1038/s41551-021-00754-5.

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Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.

Item Type:Article
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URLURL TypeDescription ReadCube access CentralDiscussion Paper Paper ItemSource data for all figures ItemOpen COVID License ItemVideos of the workflow for SwabSeq ItemR package ItemCodes for primer design
Bloom, Joshua S.0000-0002-7241-1648
Sathe, Laila0000-0003-1016-3295
Gasperini, Molly0000-0003-4559-8432
Lubock, Nathan B.0000-0001-8064-2465
Marquette, Dawn0000-0002-3964-7683
Booeshaghi, A. Sina0000-0002-6442-4502
Brandenberg, Oliver F.0000-0001-5662-1234
Guo, Longhua0000-0001-9690-9750
Boocock, James0000-0003-0323-8818
Simpkins, Scott W.0000-0002-5997-2838
Oland, Gabriel0000-0002-6941-3060
Butte, Manish J.0000-0002-4490-5595
Damoiseaux, Robert0000-0002-7611-7534
Cooper, Aaron R.0000-0003-4588-2513
Yin, Yi0000-0003-0963-2672
Pachter, Lior0000-0002-9164-6231
Garner, Omai B.0000-0002-7366-2692
Flint, Jonathan0000-0002-9427-4429
Eskin, Eleazar0000-0003-1149-4758
Luo, Chongyuan0000-0002-8541-0695
Kosuri, Sriram0000-0002-4661-0600
Kruglyak, Leonid0000-0002-8065-3057
Arboleda, Valerie A.0000-0002-9687-9122
Alternate Title:Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing
Additional Information:© 2021 Nature Publishing Group. Received 20 November 2020; Accepted 20 May 2021; Published 01 July 2021. We thank J. Semel for her support. We also thank the staff at the Held Foundation and the Carol Moss Foundation for their support of this project; staff at the UCLA David Geffen School of Medicine’s Dean’s Office for their support; Fast Grants Inc. for funding this work; L. Starita, B. Martin, J. Gehring, S. Srivatsan, J. Shendure and the members of the Covid Testing Scaleup Slack for their input, guidance and openness in sharing their processes; M. Berro for her guidance with the FDA EUA201963; the clinical laboratory scientists at the UCLA Clinical Microbiology laboratory for their assistance in collecting and processing the remnant specimens and data; our staff at the UCLA SwabSeq COVID19 Testing laboratory for deploying our CLIA test; and L. Yost and A. Martin for their advice and guidance during our scaling process. This work was supported by funding from the Howard Hughes Medical Institute (to L.K.) and DP5OD024579 (to V.A.A.). I.L. is supported by T32GM008042. Figures 1a,f and 3b created with Data availability: The main data supporting the results in this study are available within the paper and its Supplementary Information. Source data for all figures are available at GitHub ( All protocols and primers are available under an Open COVID License online ( Videos of the workflow for SwabSeq assay are available at Figshare ( Code availability: All code can be accessed at GitHub ( An R package to automate the diagnosis of patient samples is available at GitHub ( Codes for primer design and for the analysis of cross-reactivity can be found at GitHub ( The core technology has been made available under the Open COVID Pledge, and software and data under the MIT license (UCLA) and Apache 2.0 license (Octant). Author Contributions: J.S.B. and V.A.A. wrote the manuscript with assistance from C.L., J.F., L.K., E.E., E.M.J., A.R.C., N.B.L., M.G. and S.Kosuri. E.M.J., A.R.C., N.B.L., M.G., S.W.S., J.S.B. and S.Kosuri designed barcodes and performed early testing and analysis of protocols and reagents. C.L., Y.Y., Y.Z., L.G., R.D. and M.J.B. provided early guidance and key automation resources. E.E., D.H., N.L. and C.K. developed the registration webapp and IT infrastructure. L.S., C.M., M.G., E.M.J., N.B.L., S.Kosuri, I.L., O.F.B., V.A.A. and J.S.B. performed and analysed experiments. A.S.B. and L.P. analysed misassignment of index barcodes. V.A.A., O.B.G., S.C., E.E.H., G.O. and B.J.C. collected and processed clinical samples. D.M. optimized operational protocols and D.M., S.Kay, M.L., T.L. and E.E. optimized scale up. E.E., L.K., J.F., C.L., Y.Y., Y.Z. and J.B. provided helpful insights into protocols, software, and development and optimization of our specimen collection and handling. F.Y., E.M.T., K.M.K., J.P. and M.H. developed the diversified S standard mixture, N1 primers and flu primers. Competing interests: E.M.J., M.G., N.B.L., S.W.S., F.Y., E.M.T., K.M.K., J.P. and S.Kosuri are employed by and hold equity in Octant Inc., J.S.B. consults for and holds equity in Octant Inc., and A.R.C. holds equity in Octant Inc, which initially developed SwabSeq and has filed for patents for some of the work here, although they have been made available under the Open COVID License ( Peer review information: Nature Biomedical Engineering thanks Enzo Poirier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Funding AgencyGrant Number
Held FoundationUNSPECIFIED
Carol Moss FoundationUNSPECIFIED
Fast Grants, Inc.UNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH Predoctoral FellowshipT32GM008042
Issue or Number:7
PubMed Central ID:PMC7480060
Record Number:CaltechAUTHORS:20201119-132151980
Persistent URL:
Official Citation:Bloom, J.S., Sathe, L., Munugala, C. et al. Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples. Nat Biomed Eng 5, 657–665 (2021).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:106738
Deposited By: Tony Diaz
Deposited On:19 Nov 2020 22:33
Last Modified:26 Jul 2021 22:18

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