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Article ; Online: Protocol for neurogenin-2-mediated induction of human stem cell-derived neural progenitor cells.

Guss, Ellen J / Sathe, Laila / Dai, Alexander / Derebenskiy, Tim / Vega, Ana Rodriguez / Eggan, Kevin / Wells, Michael F

STAR protocols

2024 Volume 5, Issue 1, Page(s) 102878

Abstract: Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here, we present a protocol to generate NPCs rapidly and reproducibly from human stem ... ...

Abstract	Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here, we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells-termed stem cell-derived Ngn2-accelerated progenitor cells-followed by steps for expansion, purification, banking, and quality assessment. For complete details on the use and execution of this protocol, please refer to Wells et al.
MeSH term(s)	Humans ; Mice ; Animals ; Cell Differentiation/physiology ; Neural Stem Cells ; Pluripotent Stem Cells
Language	English
Publishing date	2024-02-08
Publishing country	United States
Document type	Journal Article
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2024.102878
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Article ; Online: Lean Principles to Improve Quality in High-Throughput COVID-19 Testing Using SwabSeq: A Barcoded Sequencing-Based Testing Platform.

Jones, Janae / Saul, Rosita / Sathe, Laila / Xie, Joanna / Marquette, Dawn / Arboleda, Valerie A

Laboratory medicine

2021 Volume 53, Issue 1, Page(s) e8–e13

Abstract: Objective: To describe and quantify the effect of quality control (QC) metrics to increase testing efficiency in a high-complexity, Clinical Laboratory Improvement Amendments-certified laboratory that uses amplicon-based, next generation sequencing for ... ...

Abstract	Objective: To describe and quantify the effect of quality control (QC) metrics to increase testing efficiency in a high-complexity, Clinical Laboratory Improvement Amendments-certified laboratory that uses amplicon-based, next generation sequencing for the clinical detection of SARS-CoV-2. To enable rapid scalability to several thousands of specimens per day without fully automated platforms, we developed internal QC methods to ensure high-accuracy testing. Methods: We implemented procedures to increase efficiency by applying the Lean Six Sigma model into our sequencing-based COVID-19 detection. Results: The application of the Lean Six Sigma model increased laboratory efficiency by reducing errors, allowing for a higher testing volume to be met with minimal staffing. Furthermore, these improvements resulted in an improved turnaround time. Conclusion: Lean Six Sigma model execution has increased laboratory efficiency by decreasing critical testing errors and has prepared the laboratory for future scaling up to 50,000 tests per day.
MeSH term(s)	COVID-19/diagnosis ; COVID-19 Testing ; Humans ; Laboratories, Clinical ; SARS-CoV-2/isolation & purification ; Total Quality Management
Language	English
Publishing date	2021-08-25
Publishing country	England
Document type	Journal Article
ZDB-ID	391758-7
ISSN	1943-7730 ; 0007-5027
ISSN (online)	1943-7730
ISSN	0007-5027
DOI	10.1093/labmed/lmab069
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Article ; Online: 3D Printing as an Effective Quality Assurance Implementation in Massive-Scale SARS-CoV-2 Testing at a SwabSeq Next-Generation Sequencing Laboratory.

Sathe, Laila M / Khan, Nishrat N / Williams, Jazmine M / Saul, Rosita / Jajieh, Kane / Sartippour, Maryam R / Young, Rachel / Xie, Joanna / Marquette, Dawn M / Duncan, Tiffany / Eskin, Eleazar / Arboleda, Valerie A

Laboratory medicine

2023 Volume 54, Issue 5, Page(s) 512–518

Abstract: Massive-scale SARS-CoV-2 testing using the SwabSeq diagnostic platform came with quality assurance challenges due to the novelty and scale of sequencing-based testing. The SwabSeq platform relies on accurate mapping between specimen identifiers and ... ...

Abstract	Massive-scale SARS-CoV-2 testing using the SwabSeq diagnostic platform came with quality assurance challenges due to the novelty and scale of sequencing-based testing. The SwabSeq platform relies on accurate mapping between specimen identifiers and molecular barcodes to match a result back to a patient specimen. To identify and mitigate mapping errors, we instituted quality control using placement of negative controls within a rack of patient samples. We designed 2-dimensional paper templates to fit over a 96-position rack of specimens with holes to show the control tube placements. We designed and 3-dimensionally printed plastic templates that fit onto 4 racks of patient specimens and provide accurate indications of the correct control tube placements. The final plastic templates dramatically reduced plate mapping errors from 22.55% in January 2021 to less than 1% after implementation and training in January 2021. We show how 3D printing can be a cost-effective quality assurance tool to mitigate human error in the clinical laboratory.
MeSH term(s)	Humans ; SARS-CoV-2/genetics ; COVID-19/diagnosis ; COVID-19 Testing ; High-Throughput Nucleotide Sequencing ; Printing, Three-Dimensional ; Plastics
Chemical Substances	Plastics
Language	English
Publishing date	2023-02-22
Publishing country	England
Document type	Journal Article
ZDB-ID	391758-7
ISSN	1943-7730 ; 0007-5027
ISSN (online)	1943-7730
ISSN	0007-5027
DOI	10.1093/labmed/lmac161
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Article ; Online: Genomic epidemiology of the Los Angeles COVID-19 outbreak and the early history of the B.1.43 strain in the USA.

Guo, Longhua / Boocock, James / Hilt, Evann E / Chandrasekaran, Sukantha / Zhang, Yi / Munugala, Chetan / Sathe, Laila / Alexander, Noah / Arboleda, Valerie A / Flint, Jonathan / Eskin, Eleazar / Luo, Chongyuan / Yang, Shangxin / Garner, Omai B / Yin, Yi / Bloom, Joshua S / Kruglyak, Leonid

BMC genomics

2022 Volume 23, Issue 1, Page(s) 260

Abstract: Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption of human health and activity. Being able to trace the early outbreak of SARS-CoV-2 within a locality can inform public health measures and provide ... ...

Abstract	Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption of human health and activity. Being able to trace the early outbreak of SARS-CoV-2 within a locality can inform public health measures and provide insights to contain or prevent viral transmission. Investigation of the transmission history requires efficient sequencing methods and analytic strategies, which can be generally useful in the study of viral outbreaks. Methods: The County of Los Angeles (hereafter, LA County) sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history, we carried out surveillance viral genome sequencing to determine 142 viral genomes from unique patients seeking care at the University of California, Los Angeles (UCLA) Health System. 86 of these genomes were from samples collected before April 19, 2020. Results: We found that the early outbreak in LA County, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a USA-specific strain, B.1.43, which was found predominantly in California and Washington State. While samples from LA County carried the ancestral B.1.43 genome, viral genomes from neighboring counties in California and from counties in Washington State carried additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but might have been undermined by the many introductions of SARS-CoV-2 into the region. Conclusion: Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the USA to have coordinated inter-state responses to the pandemic.
MeSH term(s)	COVID-19/epidemiology ; Disease Outbreaks ; Genomics ; Humans ; Los Angeles/epidemiology ; SARS-CoV-2/genetics
Language	English
Publishing date	2022-04-04
Publishing country	England
Document type	Journal Article
ZDB-ID	2041499-7
ISSN	1471-2164 ; 1471-2164
ISSN (online)	1471-2164
ISSN	1471-2164
DOI	10.1186/s12864-022-08488-7
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Article ; Online: Retrospective Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Symptomatic Patients Prior to Widespread Diagnostic Testing in Southern California.

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America

2021 Volume 74, Issue 2, Page(s) 271–277

Abstract: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, ...

Abstract	Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited. Methods: We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples. Results: We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January. Conclusions: We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.
MeSH term(s)	COVID-19 ; Diagnostic Techniques and Procedures ; Humans ; Los Angeles/epidemiology ; Retrospective Studies ; SARS-CoV-2
Language	English
Publishing date	2021-05-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	1099781-7
ISSN	1537-6591 ; 1058-4838
ISSN (online)	1537-6591
ISSN	1058-4838
DOI	10.1093/cid/ciab360
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Article ; Online: Genomic epidemiology of the Los Angeles COVID-19 outbreak

Guo, Longhua / Boocock, James / Hilt, Evann / Chandrasekaran, Sukantha / Zhang, Yi / Munugala, Chetan / Sathe, Laila / Alexander, Noah / Arboleda, Valerie A. / Flint, Jonathan / Eskin, Eleazar / Luo, Chongyuan / Yang, Shangxin / Garner, Omai B / Yin, Yi / Bloom, Joshua S / Kruglyak, Leonid

medRxiv

Abstract: Los Angeles (LA) County has sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history of SARS-CoV-2 in LA County, we sequenced 142 viral genomes from unique patients seeking care ... ...

Abstract	Los Angeles (LA) County has sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history of SARS-CoV-2 in LA County, we sequenced 142 viral genomes from unique patients seeking care at UCLA Health System. 86 of these genomes are from samples collected before April 19, 2020. We found that the early outbreak in LA, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a US-specific strain, B.1.43, which has been found predominantly in California and Washington State. While samples from LA County carry the ancestral B.1.43 genome, viral genomes from neighbouring counties in California and from counties in Washington State carry additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but may have been undermined by the many introductions of SARS-CoV-2 into the region. Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the U.S. to have coordinated inter-state responses to the pandemic.
Keywords	covid19
Language	English
Publishing date	2020-09-18
Publisher	Cold Spring Harbor Laboratory Press
Document type	Article ; Online
DOI	10.1101/2020.09.15.20194712
Database	COVID19

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Article ; Online: Rapid cost-effective viral genome sequencing by V-seq

Guo, Longhua / Boocock, James / Tome, Jacob M. / Chandrasekaran, Sukantha / Hilt, Evann E. / Zhang, Yi / Sathe, Laila / Li, Xinmin / Luo, Chongyuan / Kosuri, Sriram / Shendure, Jay A. / Arboleda, Valerie A. / Flint, Jonathan / Eskin, Eleazar / Garner, Omai B. / Yang, Shangxin / Bloom, Joshua S. / Kruglyak, Leonid / Yin, Yi

bioRxiv

Abstract: Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, ... ...

Abstract	Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3’ end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.
Keywords	covid19
Publisher	BioRxiv
Document type	Article ; Online
DOI	10.1101/2020.08.15.252510
Database	COVID19

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Article ; Online: Rapid cost-effective viral genome sequencing by V-seq

Guo, Longhua / Boocock, James / Tome, Jacob / Chandrasekaran, Sukantha / Hilt, Evann / Zhang, Yi / Sathe, Laila / Li, Xinmin / Luo, Chongyuan / Kosuri, Sriram / Shendure, Jay / Arboleda, Valerie / Flint, Jonathan / Eskin, Eleazar / Garner, Omai / Yang, Shangxin / Bloom, Joshua / Kruglyak, Leonid / Yin, Yi

2020

Abstract: ABSTRACT Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are ... ...

Abstract	ABSTRACT Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3’ end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.
Keywords	covid19
Publishing date	2020-08-15
Publisher	eScholarship, University of California
Publishing country	us
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing.

Bloom, Joshua S / Sathe, Laila / Munugala, Chetan / Jones, Eric M / Gasperini, Molly / Lubock, Nathan B / Yarza, Fauna / Thompson, Erin M / Kovary, Kyle M / Park, Jimin / Marquette, Dawn / Kay, Stephania / Lucas, Mark / Love, TreQuan / Booeshaghi, A Sina / Brandenberg, Oliver F / Guo, Longhua / Boocock, James / Hochman, Myles /

medRxiv : the preprint server for health sciences

2021

Abstract: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of ... ...

Abstract	The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission
Keywords	covid19
Language	English
Publishing date	2021-03-09
Publishing country	United States
Document type	Preprint
DOI	10.1101/2020.08.04.20167874
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Article ; Online: Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples.

Bloom, Joshua S / Sathe, Laila / Munugala, Chetan / Jones, Eric M / Gasperini, Molly / Lubock, Nathan B / Yarza, Fauna / Thompson, Erin M / Kovary, Kyle M / Park, Jimin / Marquette, Dawn / Kay, Stephania / Lucas, Mark / Love, TreQuan / Sina Booeshaghi, A / Brandenberg, Oliver F / Guo, Longhua / Boocock, James / Hochman, Myles /

Nature biomedical engineering

2021 Volume 5, Issue 7, Page(s) 657–665

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	High-Throughput Nucleotide Sequencing ; Humans ; RNA, Viral/genetics ; SARS-CoV-2/genetics ; SARS-CoV-2/pathogenicity ; Saliva/virology ; Sensitivity and Specificity
Chemical Substances	RNA, Viral
Language	English
Publishing date	2021-07-01
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	2157-846X
ISSN (online)	2157-846X
DOI	10.1038/s41551-021-00754-5
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