Difference between revisions of "Journal:Comprehensive analyses of SARS-CoV-2 transmission in a public health virology laboratory"

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==Introduction==
==Introduction==
[[SARS-CoV-2]] (severe acute respiratory syndrome coronavirus 2) is a novel [[coronavirus]] that emerged in Wuhan, China in December 2019<ref name="LuOut20">{{cite journal |title=Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle |journal=Journal of Medical Virology |author=Lu, H.; Stratton, C.W.; Tang, Y.-W. |volume=92 |issue=4 |pages=401–2 |year=2020 |doi=10.1002/jmv.25678 |pmid=31950516 |pmc=PMC7166628}}</ref> and has rapidly spread across China and to many countries worldwide, causing severe respiratory disease leading to substantial morbidity and mortality.<ref name="LiEarly20">{{cite journal |title=Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia |journal=The New England Journal of Medicine |author=Li, Q.; Guan, X.; Wu, P. et al. |year=2020 |doi=10.1056/NEJMoa2001316 |pmid=31995857}}</ref><ref name="WangANovel20">{{cite journal |title=A novel coronavirus outbreak of global health concern |journal=Lancet |author=Wang, C.; Horby, P.W.; Hayden, F.G. et al. |volume=395 |issue=10223 |pages=470–73 |year=2020 |doi=10.1016/S0140-6736(20)30185-9 |pmid=31986257 |pmc=PMC7135038}}</ref><ref name="HolshueFirst20">{{cite journal |title=First Case of 2019 Novel Coronavirus in the United States |journal=New England Journal of Medicine |author=Holshue, M.L.; DeBolt, C.; Lindquist, S. et al. |volume=382 |issue=10 |pages=929–36 |year=2020 |doi=10.1056/NEJMoa2001191 |pmid=32004427 |pmc=PMC7092802}}</ref><ref name="BajemaPersons20">{{cite journal |title=Persons Evaluated for 2019 Novel Coronavirus - United States, January 2020 |journal=Morbidity and Mortality Weekly Report |author=Bajema, K.L.; Oster, A.M.; McGovern, O.L. et al. |volume=69 |issue=6 |pages=166–70 |year=2020 |doi=10.15585/mmwr.mm6906e1 |pmid=32053579 |pmc=PMC7017962}}</ref><ref name="CDCCOVIDView">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/covid-data/covidview/index.html |title=COVIDView: A Weekly Surveillance Summary of U.S. COVID-19 Activity |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |accessdate=03 July 2020}}</ref> This novel virus is a potential threat to human health worldwide and a major global health concern due to person-to-person transmission, a current lack of vaccination, and a lack of effective therapeutic options.<ref name="WangANovel20" /><ref name="WHOCorona">{{cite web |url=https://www.euro.who.int/en/health-topics/health-emergencies/coronavirus-covid-19/novel-coronavirus-2019-ncov |title=Coronavirus disease (COVID-19) pandemic |author=World Health Organization |publisher=World Health Organization |accessdate=15 July 2020}}</ref> Major SARS-CoV-2 worldwide [[Wikipedia:Clade|clades]] have been proposed by nomenclature systems, including Nextstrain<ref name="HadfieldNextstrain18">{{cite journal |title=Nextstrain: Real-time tracking of pathogen evolution |journal=Bioinformatics |author=Hadfield, J.; Megill, C.; Bell, S.M. et al. |volume=34 |issue=23 |pages=4121-4123 |year=2018 |doi=10.1093/bioinformatics/bty407 |pmid=29790939 |pmc=PMC6247931}}</ref> and the Global Initiative on Sharing All Influenza Data (GISAID).<ref name="ElbeData17">{{cite journal |title=Data, disease and diplomacy: GISAID's innovative contribution to global health |journal=Global Challenges |author=Elbe, S.; Buckland-Merrett, G. |volume=1 |issue=1 |pages=33–46 |year=2017 |doi=10.1002/gch2.1018 |pmid=31565258 |pmc=PMC6607375}}</ref> These are based on viral genomes from >57,000 sequences submitted in GISAID.<ref name="ElbeData17" /> For example, using Nextstrain’s nomenclature, there are currently five major clades: 19A (the root clade) and 19B, and clades 20A, B, and C. These are widespread in Europe and include a mutation in the spike protein, D614G, that is associated with increased infectivity and higher viral loads.<ref name="KorberTracking20">{{cite journal |title=Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus |journal=Cell |author=Korber, B.; Fischer, W.M.; Gnanakaran, S. et al. |volume=S0092-8674 |issue=20 |pages=30820-5 |year=2020 |doi=10.1016/j.cell.2020.06.043 |pmid=32697968 |pmc=PMC7332439}}</ref>
[[SARS-CoV-2]] (severe acute respiratory syndrome coronavirus 2) is a novel [[coronavirus]] that emerged in Wuhan, China in December 2019<ref name="LuOut20">{{cite journal |title=Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle |journal=Journal of Medical Virology |author=Lu, H.; Stratton, C.W.; Tang, Y.-W. |volume=92 |issue=4 |pages=401–2 |year=2020 |doi=10.1002/jmv.25678 |pmid=31950516 |pmc=PMC7166628}}</ref> and has rapidly spread across China and to many countries worldwide, causing severe respiratory disease ([[COVID-19]]) leading to substantial morbidity and mortality.<ref name="LiEarly20">{{cite journal |title=Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia |journal=The New England Journal of Medicine |author=Li, Q.; Guan, X.; Wu, P. et al. |year=2020 |doi=10.1056/NEJMoa2001316 |pmid=31995857}}</ref><ref name="WangANovel20">{{cite journal |title=A novel coronavirus outbreak of global health concern |journal=Lancet |author=Wang, C.; Horby, P.W.; Hayden, F.G. et al. |volume=395 |issue=10223 |pages=470–73 |year=2020 |doi=10.1016/S0140-6736(20)30185-9 |pmid=31986257 |pmc=PMC7135038}}</ref><ref name="HolshueFirst20">{{cite journal |title=First Case of 2019 Novel Coronavirus in the United States |journal=New England Journal of Medicine |author=Holshue, M.L.; DeBolt, C.; Lindquist, S. et al. |volume=382 |issue=10 |pages=929–36 |year=2020 |doi=10.1056/NEJMoa2001191 |pmid=32004427 |pmc=PMC7092802}}</ref><ref name="BajemaPersons20">{{cite journal |title=Persons Evaluated for 2019 Novel Coronavirus - United States, January 2020 |journal=Morbidity and Mortality Weekly Report |author=Bajema, K.L.; Oster, A.M.; McGovern, O.L. et al. |volume=69 |issue=6 |pages=166–70 |year=2020 |doi=10.15585/mmwr.mm6906e1 |pmid=32053579 |pmc=PMC7017962}}</ref><ref name="CDCCOVIDView">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/covid-data/covidview/index.html |title=COVIDView: A Weekly Surveillance Summary of U.S. COVID-19 Activity |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |accessdate=03 July 2020}}</ref> This novel virus is a potential threat to human health worldwide and a major global health concern due to person-to-person transmission, a current lack of vaccination, and a lack of effective therapeutic options.<ref name="WangANovel20" /><ref name="WHOCorona">{{cite web |url=https://www.euro.who.int/en/health-topics/health-emergencies/coronavirus-covid-19/novel-coronavirus-2019-ncov |title=Coronavirus disease (COVID-19) pandemic |author=World Health Organization |publisher=World Health Organization |accessdate=15 July 2020}}</ref> Major SARS-CoV-2 worldwide [[Wikipedia:Clade|clades]] have been proposed by nomenclature systems, including Nextstrain<ref name="HadfieldNextstrain18">{{cite journal |title=Nextstrain: Real-time tracking of pathogen evolution |journal=Bioinformatics |author=Hadfield, J.; Megill, C.; Bell, S.M. et al. |volume=34 |issue=23 |pages=4121-4123 |year=2018 |doi=10.1093/bioinformatics/bty407 |pmid=29790939 |pmc=PMC6247931}}</ref> and the Global Initiative on Sharing All Influenza Data (GISAID).<ref name="ElbeData17">{{cite journal |title=Data, disease and diplomacy: GISAID's innovative contribution to global health |journal=Global Challenges |author=Elbe, S.; Buckland-Merrett, G. |volume=1 |issue=1 |pages=33–46 |year=2017 |doi=10.1002/gch2.1018 |pmid=31565258 |pmc=PMC6607375}}</ref> These are based on viral genomes from >57,000 sequences submitted in GISAID.<ref name="ElbeData17" /> For example, using Nextstrain’s nomenclature, there are currently five major clades: 19A (the root clade) and 19B, and clades 20A, B, and C. These are widespread in Europe and include a mutation in the spike protein, D614G, that is associated with increased infectivity and higher viral loads.<ref name="KorberTracking20">{{cite journal |title=Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus |journal=Cell |author=Korber, B.; Fischer, W.M.; Gnanakaran, S. et al. |volume=S0092-8674 |issue=20 |pages=30820-5 |year=2020 |doi=10.1016/j.cell.2020.06.043 |pmid=32697968 |pmc=PMC7332439}}</ref>


Non-SARS-CoV-2 human coronaviruses have been circulating worldwide since the late 1960s.<ref name="BradburneEffect67">{{cite journal |title=Effects of a "new" human respiratory virus in volunteers |journal=British Medical Journal |author=Bradburne, A.F.; Bynoe, M.L.; Tyrrell, D.A. |volume=3 |issue=5568 |pages=767–9 |year=1967 |doi=10.1136/bmj.3.5568.767 |pmid=6043624 |pmc=PMC1843247}}</ref><ref name="LarsonIsol80">{{cite journal |title=Isolation of rhinoviruses and coronaviruses from 38 colds in adults |journal=Journal of Medical Virology |author=Larson, H.E.; Reed, S.E.; Tyrrell, D.A. |volume=5 |issue=3 |pages=221–9 |year=1980 |doi=10.1002/jmv.1890050306 |pmid=6262450 |pmc=PMC7167084}}</ref> The current rate of circulation of SARS-CoV-2 in Israel in the winter season is still unknown; however, analysis of Israeli specimens during the 2015–2016 winter season revealed that non-SARS-CoV-2 human coronaviruses circulate simultaneously with other common respiratory viruses, with 10% human coronavirus-positive cases.<ref name="FriedmanHuman18">{{cite journal |title=Human Coronavirus Infections in Israel: Epidemiology, Clinical Symptoms and Summer Seasonality of HCoV-HKU1 |journal=Viruses |author=Friedman, N.; Alter, H.; Hindiyeh, M. et al. |volume=10 |issue=10 |at=515 |year=1980 |doi=10.3390/v10100515 |pmid=30241410 |pmc=PMC6213580}}</ref>
Non-SARS-CoV-2 human coronaviruses have been circulating worldwide since the late 1960s.<ref name="BradburneEffect67">{{cite journal |title=Effects of a "new" human respiratory virus in volunteers |journal=British Medical Journal |author=Bradburne, A.F.; Bynoe, M.L.; Tyrrell, D.A. |volume=3 |issue=5568 |pages=767–9 |year=1967 |doi=10.1136/bmj.3.5568.767 |pmid=6043624 |pmc=PMC1843247}}</ref><ref name="LarsonIsol80">{{cite journal |title=Isolation of rhinoviruses and coronaviruses from 38 colds in adults |journal=Journal of Medical Virology |author=Larson, H.E.; Reed, S.E.; Tyrrell, D.A. |volume=5 |issue=3 |pages=221–9 |year=1980 |doi=10.1002/jmv.1890050306 |pmid=6262450 |pmc=PMC7167084}}</ref> The current rate of circulation of SARS-CoV-2 in Israel in the winter season is still unknown; however, analysis of Israeli specimens during the 2015–2016 winter season revealed that non-SARS-CoV-2 human coronaviruses circulate simultaneously with other common respiratory viruses, with 10% human coronavirus-positive cases.<ref name="FriedmanHuman18">{{cite journal |title=Human Coronavirus Infections in Israel: Epidemiology, Clinical Symptoms and Summer Seasonality of HCoV-HKU1 |journal=Viruses |author=Friedman, N.; Alter, H.; Hindiyeh, M. et al. |volume=10 |issue=10 |at=515 |year=1980 |doi=10.3390/v10100515 |pmid=30241410 |pmc=PMC6213580}}</ref>
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SARS-CoV-2 circulation in the general population in Israel and worldwide is being assessed using [[Polymerase chain reaction#Variations|real-time polymerase chain reaction]] (qPCR). A rapid development of qPCR diagnostic tests specific for SARS-CoV-2 genes has enabled fast and accurate laboratory tests for suspected individuals.<ref name="CormanDetect20">{{cite journal |title=Detection of 2019 Novel Coronavirus (2019-nCoV) by Real-Time RT-PCR |journal=Euro Surveillance |author=Corman, V.M.; Landt, O.; Kaiser, M. et al. |volume=25 |issue=3 |at=2000045 |year=2020 |doi=10.2807/1560-7917.ES.2020.25.3.2000045 |pmid=31992387 |pmc=PMC6988269}}</ref> These tests were successfully evaluated in Israel’s central virology laboratory (ICVL), where SARS-CoV-2 suspected specimens were exclusively examined, starting from the first importation case of SARS-CoV-2 into Israel at the end of February until the middle of March 2020. Starting with the first suspected case in Israel, all specimens received in ICVL facilities were dealt with using the strictest safety directions and [[Biosafetly level#Levels|biosafety level 2 (BSL2) or greater safety conditions.<ref name="WHOLab13">{{cite web |url=https://www.who.int/csr/disease/coronavirus_infections/Biosafety_InterimRecommendations_NovelCoronavirus_19Feb13.pdf |format=PDF |title=Laboratory biorisk management for laboratories handling human specimens suspected or confirmed to contain novel coronavirus: Interim recommendations |author=World Health Organization |publisher=World Health Organization |date=19 February 2013 |accessdate=11 May 2020}}</ref><ref name="CDCInterim20">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html |title=Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19) |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |accessdate=11 May 2020}}</ref> Until mid-March 2020, all SARS-CoV-2 positive cases in Israel were isolated in a designated quarantine facility; however, physical distancing and mandatory mask-wearing were not customary or enforced at that time in Israel.
SARS-CoV-2 circulation in the general population in Israel and worldwide is being assessed using [[Polymerase chain reaction#Variations|real-time polymerase chain reaction]] (qPCR). A rapid development of qPCR diagnostic tests specific for SARS-CoV-2 genes has enabled fast and accurate laboratory tests for suspected individuals.<ref name="CormanDetect20">{{cite journal |title=Detection of 2019 Novel Coronavirus (2019-nCoV) by Real-Time RT-PCR |journal=Euro Surveillance |author=Corman, V.M.; Landt, O.; Kaiser, M. et al. |volume=25 |issue=3 |at=2000045 |year=2020 |doi=10.2807/1560-7917.ES.2020.25.3.2000045 |pmid=31992387 |pmc=PMC6988269}}</ref> These tests were successfully evaluated in Israel’s central virology laboratory (ICVL), where SARS-CoV-2 suspected specimens were exclusively examined, starting from the first importation case of SARS-CoV-2 into Israel at the end of February until the middle of March 2020. Starting with the first suspected case in Israel, all specimens received in ICVL facilities were dealt with using the strictest safety directions and [[Biosafetly level#Levels|biosafety level 2 (BSL2) or greater safety conditions.<ref name="WHOLab13">{{cite web |url=https://www.who.int/csr/disease/coronavirus_infections/Biosafety_InterimRecommendations_NovelCoronavirus_19Feb13.pdf |format=PDF |title=Laboratory biorisk management for laboratories handling human specimens suspected or confirmed to contain novel coronavirus: Interim recommendations |author=World Health Organization |publisher=World Health Organization |date=19 February 2013 |accessdate=11 May 2020}}</ref><ref name="CDCInterim20">{{cite web |url=https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html |title=Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19) |author=Centers for Disease Control and Prevention |publisher=Centers for Disease Control and Prevention |accessdate=11 May 2020}}</ref> Until mid-March 2020, all SARS-CoV-2 positive cases in Israel were isolated in a designated quarantine facility; however, physical distancing and mandatory mask-wearing were not customary or enforced at that time in Israel.


In mid-March 2020, several cases of SARS-CoV-2 infection were identified in ICVL, some of which probably originated from an infected worker, as speculated by the inquiry-based [[Epidemiology|epidemiological]] investigation. SARS-CoV-2 airborne transmission was demonstrated to be the most efficient among all transmission routes<ref name="LoftiCOVID20">{{cite journal |title=COVID-19: Transmission, prevention, and potential therapeutic opportunities |journal=Clinica Chimica Acta |author=Lotfi, M.; Hamblin, M.R.; Rezaei, N. |volume=508 |pages=254–66 |year=2020 |doi=10.1016/j.cca.2020.05.044 |pmid=32474009 |pmc=PMC7256510}}</ref><ref name="LoftiCOVID20">{{cite journal |title=Identifying airborne transmission as the dominant route for the spread of COVID-19 |journal=Proceedings of the National Academy of Sciences of the United States of America |author=Zhang, R.; Li, Y.; Zhang, A.L. et al. |volume=117 |issue=26 |pages=14857-14863 |year=2020 |doi=10.1073/pnas.2009637117 |pmid=32527856 |pmc=PMC7334447}}</ref>, and contagious even in the pre-symptomatic stages<ref name="LaiAsymp20">{{cite journal |title=Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and myths |journal=Journal of Microbiology, Immunology, and Infection |author=Lai, C.-C.; Liu, Y.H.; Wang, C,-Y. et al. |volume=53 |issue=3 |pages=404–12 |year=2020 |doi=10.1016/j.jmii.2020.02.012 |pmid=32173241 |pmc=PMC7128959}}</ref><ref name="RotheTransm20">{{cite journal |title=Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany |journal=New England Journal of Medicine |author=Rothe, C.; Schunk, M.; Sothmann, P. et al. |volume=382 |issue=10 |pages=970-971 |year=2020 |doi=10.1056/NEJMc2001468 |pmid=32003551 |pmc=PMC7120970}}</ref>, such that silent virus spread easily occurs. Infection at workplaces was shown as a common transmission route in Israel in the early stages of the virus' spread, probably facilitated, in the case of the ICVL outbreak, by crowded workspaces and lack of social distancing and mask wearing at that time.
In mid-March 2020, several cases of SARS-CoV-2 infection were identified in ICVL, some of which probably originated from an infected worker, as speculated by the inquiry-based [[Epidemiology|epidemiological]] investigation. SARS-CoV-2 airborne transmission was demonstrated to be the most efficient among all transmission routes<ref name="LoftiCOVID20">{{cite journal |title=COVID-19: Transmission, prevention, and potential therapeutic opportunities |journal=Clinica Chimica Acta |author=Lotfi, M.; Hamblin, M.R.; Rezaei, N. |volume=508 |pages=254–66 |year=2020 |doi=10.1016/j.cca.2020.05.044 |pmid=32474009 |pmc=PMC7256510}}</ref><ref name="ZhangIdent20">{{cite journal |title=Identifying airborne transmission as the dominant route for the spread of COVID-19 |journal=Proceedings of the National Academy of Sciences of the United States of America |author=Zhang, R.; Li, Y.; Zhang, A.L. et al. |volume=117 |issue=26 |pages=14857-14863 |year=2020 |doi=10.1073/pnas.2009637117 |pmid=32527856 |pmc=PMC7334447}}</ref>, and contagious even in the pre-symptomatic stages<ref name="LaiAsymp20">{{cite journal |title=Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and myths |journal=Journal of Microbiology, Immunology, and Infection |author=Lai, C.-C.; Liu, Y.H.; Wang, C,-Y. et al. |volume=53 |issue=3 |pages=404–12 |year=2020 |doi=10.1016/j.jmii.2020.02.012 |pmid=32173241 |pmc=PMC7128959}}</ref><ref name="RotheTransm20">{{cite journal |title=Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany |journal=New England Journal of Medicine |author=Rothe, C.; Schunk, M.; Sothmann, P. et al. |volume=382 |issue=10 |pages=970-971 |year=2020 |doi=10.1056/NEJMc2001468 |pmid=32003551 |pmc=PMC7120970}}</ref>, such that silent virus spread easily occurs. Infection at workplaces was shown as a common transmission route in Israel in the early stages of the virus' spread, probably facilitated, in the case of the ICVL outbreak, by crowded workspaces and lack of social distancing and mask wearing at that time.


This study thoroughly investigates the SARS-CoV-2 ICVL outbreak by examining infected ICVL workers, several epidemiologically-related family members, and work surfaces from ICVL facilities. Application of SARS-CoV-2 whole [[Genomics|genome]] [[next-generation sequencing]] (NGS), qPCR, serology testing, and phylogenetic tree analyses elucidate person-to-person transmission events, map individual and common mutations, and examine suspicions regarding contaminated surfaces. This study demonstrates the added value of molecular epidemiology based on complete viral genomes in elucidating person-to-person transmission, reveals silent infections in non-symptomatic ICVL staff members via serology testing, and confirms that the strict safety regulations observed in ICVL most likely prevented further spread of the virus.
This study thoroughly investigates the SARS-CoV-2 ICVL outbreak by examining infected ICVL workers, several epidemiologically-related family members, and work surfaces from ICVL facilities. Application of SARS-CoV-2 whole [[Genomics|genome]] [[next-generation sequencing]] (NGS), qPCR, serology testing, and phylogenetic tree analyses elucidate person-to-person transmission events, map individual and common mutations, and examine suspicions regarding contaminated surfaces. This study demonstrates the added value of molecular epidemiology based on complete viral genomes in elucidating person-to-person transmission, reveals silent infections in non-symptomatic ICVL staff members via serology testing, and confirms that the strict safety regulations observed in ICVL most likely prevented further spread of the virus.
==Materials and methods==
===Sample collection, nucleic acid extraction, and viral genome quantification by qPCR===
Immediately following the identification of the first ICVL infection case (S1) on March 15, 2020, [[nasopharyngeal swab]]s from all 56 ICVL staff members and another ten non-ICVL staff who worked at the lab around this time were collected, most of them on the same day and a few on the next day. This comprehensive screening test was performed only once. Additional tests for ICVL staff were conducted for a symptomatic individual (''n'' = 1), symptomatic relatives (''n'' = 2), and for essential workers who were required to work at the laboratory (''n'' = 2). Viral genomes were extracted from 200 µL respiratory specimens with the MagNA PURE 96 (Roche, Mannheim, Germany) according to the manufacturer instructions, and real-time (or quantitative) [[reverse transcription polymerase chain reaction]] (qRT-PCR) reactions using primers corresponding to the SARS-CoV-2 envelope (E) gene were performed as previously described by Corman ''et al.''<ref name="CormanDetect20" /> All samples were tested for the human RNAseP gene, which served as a housekeeping gene. The qRT-PCR reactions were performed in 25 µL Ambion Ag-Path Master Mix (Life Technologies, Carlsbad, CA, USA) using TaqMan Chemistry on the ABI 7500 instrument. Nucleic extraction samples from SARS-CoV-2-positive staff members (S1–S6) and related family members (S7—S4′s spouse and S8—S3′s spouse) were taken for further molecular analysis.
===Specific amplification of SARS-CoV-2 from clinical samples===
RNA in extracted nucleic acids was reverse transcribed to single strand cDNA using SuperScript IV (ThermoFisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions. SARS-CoV-2-specific primers designed to capture SARS-CoV-2 whole genome (version 1—total 218 primers, divided into two primer pools designed by Josh Quick from ARTIC Network) were used to generate double strand cDNA and amplify it via PCR using Q5 Hot Start DNA Polymerase (NEB).<ref name="ANSARS20">{{cite web |url=https://artic.network/ncov-2019 |title=SARS-CoV-2 |author=Arctic Network |date=24 March 2020 |accessdate=15 July 2020}}</ref> Briefly, each sample underwent two PCR reactions with primer pool 1 or 2 and 5X Q5 reaction buffer, 19 mM dNTPs and nuclease-free water. Resulting DNA was combined and quantified with the Qubit dsDNA BR Assay kit (ThermoFisher Scientific) as per manufacturer’s instructions, and 1ng of amplicon DNA in 5 µL per sample was taken into library preparation.
===Library preparation and sequencing===
Libraries were prepared using the NexteraXT library preparation kit and NexteraXT index kit V2 as per manufacturer’s instructions (Illumina, San Diego, CA, USA). Libraries were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA), and library concentration was measured by Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Library validation and mean fragment size was determined by TapeStation 4200 via DNA HS D1000 kit (Agilent, Santa Clara, CA, USA). The mean fragment size was ~400 bp, as expected. The library mean fragment size and concentration molarity was calculated and each library was diluted to 4 nM. Libraries were pooled, denatured, and diluted to 10pM and sequenced on MiSeq with V3 2X300 bp run kit (Illumina). Sequences are available in GISAID (accession numbers: EPI_ISL_435284, EPI_ISL_435286, EPI_ISL435287, EPI_ISL435289, EPI_ISL435291, EPI_ISL_435292, EPI_ISL447250, EPI_ISL447251).
===[[Bioinformatics]] analyses===
The fastq files were subjected to quality control using FastQC<ref name="AndrewsFastQC19">{{cite web |url=http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ |title=FastQC |author=Andrews, S. |work=Babraham Bioinformatics |date=08 January 2019}}</ref> and MultiQC<ref name="EwelsMultiQC16">{{cite journal |title=MultiQC: Summarize analysis results for multiple tools and samples in a single report |journal=Bioinformatics |author=Ewals, P.; Magnusson, M.; Lundin, S. et al. |volume=32 |issue=19 |pages=3047-8 |year=2016 |doi=10.1093/bioinformatics/btw354 |pmid=27312411 |pmc=PMC5039924}}</ref>, and low-quality sequences were filtered using trimmomatic.<ref name="BolgerTrimmo14">{{cite journal |title=Trimmomatic: A flexible trimmer for Illumina sequence data |journal=Bioinformatics |author=Bolger, A.M.; Lohse, M.; Usadel, B. et al. |volume=30 |issue=15 |pages=2114-20 |year=2014 |doi=10.1093/bioinformatics/btu170 |pmid=24695404 |pmc=PMC4103590}}</ref> To obtain a consensus sequence per sample, paired-end fastq files were combined for each sample via the Unix <code>cat</code> command. SARS-CoV-2 reference genome was downloaded from the national center for biotechnology information (NCBI) (NC_045512.2) and indexed using Burrows-Wheeler aligner (BWA).<ref name="LiFast10">{{cite journal |title=Fast and accurate long-read alignment with Burrows-Wheeler transform |journal=Bioinformatics |author=Li, H.; Durbin, R. |volume=26 |issue=5 |pages=589–95 |year=2010 |doi=10.1093/bioinformatics/btp698 |pmid=20080505 |pmc=PMC2828108}}</ref> Combined fastq files were mapped to the indexed reference genome using BWA mem.<ref name="LiFast10" /> SAMtools suite<ref name="LiTheSeq09">{{cite journal |title=The Sequence Alignment/Map format and SAMtools |journal=Bioinformatics |author=Li, H.; Handsaker, B.; Wysoker, A. et al. |volume=25 |issue=16 |pages=2078-9 |year=2009 |doi=10.1093/bioinformatics/btp352 |pmid=19505943 |pmc=PMC2723002}}</ref> was used to convert sam to bam files, remove duplicates, and filter unmapped reads. The bam files were sorted, indexed, and subjected to quality control using SAMtools suite. Coverage and depth of [[sequencing]] was calculated from sorted bam files using a custom Perl script. Integrative genome viewer (IGV) was used to observe sequencing coverage per position along the genome.<ref name="RobinsonInteg11">{{cite journal |title=Integrative genomics viewer |journal=Nature Biotechnology |author=Robinson, J.T.; Thorvaldsdóttir, H.; Winckler, W. et al. |volume=29 |issue=1 |pages=24–6 |year=2011 |doi=10.1038/nbt.1754 |pmid=21221095 |pmc=PMC3346182}}</ref> A consensus sequence was constructed for each sample using SAMtools mpileup and bcf tools<ref name="NarasimhanBCFtools16">{{cite journal |title=BCFtools/RoH: A hidden Markov model approach for detecting autozygosity from next-generation sequencing data |author=Narasimhan, V.; Danecek, P.; Scally, A. et al. |volume=32 |issue=11 |pages=1749-51 |year=2016 |doi=10.1093/bioinformatics/btw044 |pmid=26826718 |pmc=PMC4892413}}</ref> and converted to a fasta file using seqtk.<ref name="GHseqtk">{{cite web |url=https://github.com/lh3/seqtk |work=GitHub |date=2020}}</ref>


==References==
==References==

Revision as of 20:32, 17 August 2020

Full article title Comprehensive analyses of SARS-CoV-2 transmission in a public health virology laboratory
Journal Viruses
Author(s) Zuckerman, Neta S.; Pando, Rakafet; Bucris, Efrat; Drori, Yaron; Lustig, Yaniv; Erster, Oran;
Mor, Orna; Mendelson, Ella; Mandelboim, Michael
Author affiliation(s) Chaim Sheba Medical Center, Israel Ministry of Health, Tel-Aviv University
Primary contact Email: michalman at sheba dot health dot gov dot il
Year published 2020
Volume and issue 12(8)
Article # 854
DOI 10.3390/v12080854
ISSN 1999-4915
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/1999-4915/12/8/854/htm
Download https://www.mdpi.com/1999-4915/12/8/854/pdf (PDF)
Emblem-important-yellow.svg This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed.

Abstract

SARS-CoV-2 has become a major global concern as of December 2019, particularly affecting healthcare workers. As person-to-person transmission is airborne, crowded closed spaces have had high potential for rapid virus spread, especially early in the pandemic when social distancing and mask wearing were not mandatory. This retrospective study thoroughly investigates a small-scale SARS-CoV-2 outbreak in Israel’s central virology laboratory (ICVL) in mid-March 2020, in which six staff members and two related family members were infected. Suspicions regarding infection by contaminated surfaces in ICVL facilities were nullified by the negative results of a SARS-CoV-2 real-time polymerase chain reaction (qPCR) analysis of swiped work surface samples. Complete SARS-CoV-2 genomes were sequenced, and mutation analyses showed inclusion of all samples to clades 20B and 20C, possessing the spike mutation D614G. Phylogenetic analysis clarified transmission events, confirming S1 as having infected at least three other staff members while refuting the association of a staff member’s infected spouse with the ICVL transmission cluster. Finally, serology tests exhibited IgG and IgA antibodies in all infected individuals and revealed the occurrence of asymptomatic infections in additional staff members. This study demonstrates the advantages of molecular epidemiology in elucidating transmission events and exemplifies the importance of good laboratory practice, physical distancing, and mask wearing in preventing SARS-CoV-2 spread, specifically in healthcare facilities.

Keywords: 2019-nCoV, SARS-CoV-2, COVID-19, staff, infection, next-generation sequencing (NGS)

Introduction

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus that emerged in Wuhan, China in December 2019[1] and has rapidly spread across China and to many countries worldwide, causing severe respiratory disease (COVID-19) leading to substantial morbidity and mortality.[2][3][4][5][6] This novel virus is a potential threat to human health worldwide and a major global health concern due to person-to-person transmission, a current lack of vaccination, and a lack of effective therapeutic options.[3][7] Major SARS-CoV-2 worldwide clades have been proposed by nomenclature systems, including Nextstrain[8] and the Global Initiative on Sharing All Influenza Data (GISAID).[9] These are based on viral genomes from >57,000 sequences submitted in GISAID.[9] For example, using Nextstrain’s nomenclature, there are currently five major clades: 19A (the root clade) and 19B, and clades 20A, B, and C. These are widespread in Europe and include a mutation in the spike protein, D614G, that is associated with increased infectivity and higher viral loads.[10]

Non-SARS-CoV-2 human coronaviruses have been circulating worldwide since the late 1960s.[11][12] The current rate of circulation of SARS-CoV-2 in Israel in the winter season is still unknown; however, analysis of Israeli specimens during the 2015–2016 winter season revealed that non-SARS-CoV-2 human coronaviruses circulate simultaneously with other common respiratory viruses, with 10% human coronavirus-positive cases.[13]

SARS-CoV-2 circulation in the general population in Israel and worldwide is being assessed using real-time polymerase chain reaction (qPCR). A rapid development of qPCR diagnostic tests specific for SARS-CoV-2 genes has enabled fast and accurate laboratory tests for suspected individuals.[14] These tests were successfully evaluated in Israel’s central virology laboratory (ICVL), where SARS-CoV-2 suspected specimens were exclusively examined, starting from the first importation case of SARS-CoV-2 into Israel at the end of February until the middle of March 2020. Starting with the first suspected case in Israel, all specimens received in ICVL facilities were dealt with using the strictest safety directions and [[Biosafetly level#Levels|biosafety level 2 (BSL2) or greater safety conditions.[15][16] Until mid-March 2020, all SARS-CoV-2 positive cases in Israel were isolated in a designated quarantine facility; however, physical distancing and mandatory mask-wearing were not customary or enforced at that time in Israel.

In mid-March 2020, several cases of SARS-CoV-2 infection were identified in ICVL, some of which probably originated from an infected worker, as speculated by the inquiry-based epidemiological investigation. SARS-CoV-2 airborne transmission was demonstrated to be the most efficient among all transmission routes[17][18], and contagious even in the pre-symptomatic stages[19][20], such that silent virus spread easily occurs. Infection at workplaces was shown as a common transmission route in Israel in the early stages of the virus' spread, probably facilitated, in the case of the ICVL outbreak, by crowded workspaces and lack of social distancing and mask wearing at that time.

This study thoroughly investigates the SARS-CoV-2 ICVL outbreak by examining infected ICVL workers, several epidemiologically-related family members, and work surfaces from ICVL facilities. Application of SARS-CoV-2 whole genome next-generation sequencing (NGS), qPCR, serology testing, and phylogenetic tree analyses elucidate person-to-person transmission events, map individual and common mutations, and examine suspicions regarding contaminated surfaces. This study demonstrates the added value of molecular epidemiology based on complete viral genomes in elucidating person-to-person transmission, reveals silent infections in non-symptomatic ICVL staff members via serology testing, and confirms that the strict safety regulations observed in ICVL most likely prevented further spread of the virus.

Materials and methods

Sample collection, nucleic acid extraction, and viral genome quantification by qPCR

Immediately following the identification of the first ICVL infection case (S1) on March 15, 2020, nasopharyngeal swabs from all 56 ICVL staff members and another ten non-ICVL staff who worked at the lab around this time were collected, most of them on the same day and a few on the next day. This comprehensive screening test was performed only once. Additional tests for ICVL staff were conducted for a symptomatic individual (n = 1), symptomatic relatives (n = 2), and for essential workers who were required to work at the laboratory (n = 2). Viral genomes were extracted from 200 µL respiratory specimens with the MagNA PURE 96 (Roche, Mannheim, Germany) according to the manufacturer instructions, and real-time (or quantitative) reverse transcription polymerase chain reaction (qRT-PCR) reactions using primers corresponding to the SARS-CoV-2 envelope (E) gene were performed as previously described by Corman et al.[14] All samples were tested for the human RNAseP gene, which served as a housekeeping gene. The qRT-PCR reactions were performed in 25 µL Ambion Ag-Path Master Mix (Life Technologies, Carlsbad, CA, USA) using TaqMan Chemistry on the ABI 7500 instrument. Nucleic extraction samples from SARS-CoV-2-positive staff members (S1–S6) and related family members (S7—S4′s spouse and S8—S3′s spouse) were taken for further molecular analysis.

Specific amplification of SARS-CoV-2 from clinical samples

RNA in extracted nucleic acids was reverse transcribed to single strand cDNA using SuperScript IV (ThermoFisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions. SARS-CoV-2-specific primers designed to capture SARS-CoV-2 whole genome (version 1—total 218 primers, divided into two primer pools designed by Josh Quick from ARTIC Network) were used to generate double strand cDNA and amplify it via PCR using Q5 Hot Start DNA Polymerase (NEB).[21] Briefly, each sample underwent two PCR reactions with primer pool 1 or 2 and 5X Q5 reaction buffer, 19 mM dNTPs and nuclease-free water. Resulting DNA was combined and quantified with the Qubit dsDNA BR Assay kit (ThermoFisher Scientific) as per manufacturer’s instructions, and 1ng of amplicon DNA in 5 µL per sample was taken into library preparation.

Library preparation and sequencing

Libraries were prepared using the NexteraXT library preparation kit and NexteraXT index kit V2 as per manufacturer’s instructions (Illumina, San Diego, CA, USA). Libraries were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA), and library concentration was measured by Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Library validation and mean fragment size was determined by TapeStation 4200 via DNA HS D1000 kit (Agilent, Santa Clara, CA, USA). The mean fragment size was ~400 bp, as expected. The library mean fragment size and concentration molarity was calculated and each library was diluted to 4 nM. Libraries were pooled, denatured, and diluted to 10pM and sequenced on MiSeq with V3 2X300 bp run kit (Illumina). Sequences are available in GISAID (accession numbers: EPI_ISL_435284, EPI_ISL_435286, EPI_ISL435287, EPI_ISL435289, EPI_ISL435291, EPI_ISL_435292, EPI_ISL447250, EPI_ISL447251).

Bioinformatics analyses

The fastq files were subjected to quality control using FastQC[22] and MultiQC[23], and low-quality sequences were filtered using trimmomatic.[24] To obtain a consensus sequence per sample, paired-end fastq files were combined for each sample via the Unix cat command. SARS-CoV-2 reference genome was downloaded from the national center for biotechnology information (NCBI) (NC_045512.2) and indexed using Burrows-Wheeler aligner (BWA).[25] Combined fastq files were mapped to the indexed reference genome using BWA mem.[25] SAMtools suite[26] was used to convert sam to bam files, remove duplicates, and filter unmapped reads. The bam files were sorted, indexed, and subjected to quality control using SAMtools suite. Coverage and depth of sequencing was calculated from sorted bam files using a custom Perl script. Integrative genome viewer (IGV) was used to observe sequencing coverage per position along the genome.[27] A consensus sequence was constructed for each sample using SAMtools mpileup and bcf tools[28] and converted to a fasta file using seqtk.[29]


References

  1. ↑ Lu, H.; Stratton, C.W.; Tang, Y.-W. (2020). "Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle". Journal of Medical Virology 92 (4): 401–2. doi:10.1002/jmv.25678. PMC PMC7166628. PMID 31950516. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166628. 
  2. ↑ Li, Q.; Guan, X.; Wu, P. et al. (2020). "Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia". The New England Journal of Medicine. doi:10.1056/NEJMoa2001316. PMID 31995857. 
  3. ↑ 3.0 3.1 Wang, C.; Horby, P.W.; Hayden, F.G. et al. (2020). "A novel coronavirus outbreak of global health concern". Lancet 395 (10223): 470–73. doi:10.1016/S0140-6736(20)30185-9. PMC PMC7135038. PMID 31986257. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135038. 
  4. ↑ Holshue, M.L.; DeBolt, C.; Lindquist, S. et al. (2020). "First Case of 2019 Novel Coronavirus in the United States". New England Journal of Medicine 382 (10): 929–36. doi:10.1056/NEJMoa2001191. PMC PMC7092802. PMID 32004427. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092802. 
  5. ↑ Bajema, K.L.; Oster, A.M.; McGovern, O.L. et al. (2020). "Persons Evaluated for 2019 Novel Coronavirus - United States, January 2020". Morbidity and Mortality Weekly Report 69 (6): 166–70. doi:10.15585/mmwr.mm6906e1. PMC PMC7017962. PMID 32053579. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017962. 
  6. ↑ Centers for Disease Control and Prevention. "COVIDView: A Weekly Surveillance Summary of U.S. COVID-19 Activity". Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/covid-data/covidview/index.html. Retrieved 03 July 2020. 
  7. ↑ World Health Organization. "Coronavirus disease (COVID-19) pandemic". World Health Organization. https://www.euro.who.int/en/health-topics/health-emergencies/coronavirus-covid-19/novel-coronavirus-2019-ncov. Retrieved 15 July 2020. 
  8. ↑ Hadfield, J.; Megill, C.; Bell, S.M. et al. (2018). "Nextstrain: Real-time tracking of pathogen evolution". Bioinformatics 34 (23): 4121-4123. doi:10.1093/bioinformatics/bty407. PMC PMC6247931. PMID 29790939. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247931. 
  9. ↑ 9.0 9.1 Elbe, S.; Buckland-Merrett, G. (2017). "Data, disease and diplomacy: GISAID's innovative contribution to global health". Global Challenges 1 (1): 33–46. doi:10.1002/gch2.1018. PMC PMC6607375. PMID 31565258. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607375. 
  10. ↑ Korber, B.; Fischer, W.M.; Gnanakaran, S. et al. (2020). "Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus". Cell S0092-8674 (20): 30820-5. doi:10.1016/j.cell.2020.06.043. PMC PMC7332439. PMID 32697968. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332439. 
  11. ↑ Bradburne, A.F.; Bynoe, M.L.; Tyrrell, D.A. (1967). "Effects of a "new" human respiratory virus in volunteers". British Medical Journal 3 (5568): 767–9. doi:10.1136/bmj.3.5568.767. PMC PMC1843247. PMID 6043624. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1843247. 
  12. ↑ Larson, H.E.; Reed, S.E.; Tyrrell, D.A. (1980). "Isolation of rhinoviruses and coronaviruses from 38 colds in adults". Journal of Medical Virology 5 (3): 221–9. doi:10.1002/jmv.1890050306. PMC PMC7167084. PMID 6262450. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167084. 
  13. ↑ Friedman, N.; Alter, H.; Hindiyeh, M. et al. (1980). "Human Coronavirus Infections in Israel: Epidemiology, Clinical Symptoms and Summer Seasonality of HCoV-HKU1". Viruses 10 (10): 515. doi:10.3390/v10100515. PMC PMC6213580. PMID 30241410. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213580. 
  14. ↑ 14.0 14.1 Corman, V.M.; Landt, O.; Kaiser, M. et al. (2020). "Detection of 2019 Novel Coronavirus (2019-nCoV) by Real-Time RT-PCR". Euro Surveillance 25 (3): 2000045. doi:10.2807/1560-7917.ES.2020.25.3.2000045. PMC PMC6988269. PMID 31992387. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269. 
  15. ↑ World Health Organization (19 February 2013). "Laboratory biorisk management for laboratories handling human specimens suspected or confirmed to contain novel coronavirus: Interim recommendations" (PDF). World Health Organization. https://www.who.int/csr/disease/coronavirus_infections/Biosafety_InterimRecommendations_NovelCoronavirus_19Feb13.pdf. Retrieved 11 May 2020. 
  16. ↑ Centers for Disease Control and Prevention. "Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19)". Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html. Retrieved 11 May 2020. 
  17. ↑ Lotfi, M.; Hamblin, M.R.; Rezaei, N. (2020). "COVID-19: Transmission, prevention, and potential therapeutic opportunities". Clinica Chimica Acta 508: 254–66. doi:10.1016/j.cca.2020.05.044. PMC PMC7256510. PMID 32474009. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256510. 
  18. ↑ Zhang, R.; Li, Y.; Zhang, A.L. et al. (2020). "Identifying airborne transmission as the dominant route for the spread of COVID-19". Proceedings of the National Academy of Sciences of the United States of America 117 (26): 14857-14863. doi:10.1073/pnas.2009637117. PMC PMC7334447. PMID 32527856. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334447. 
  19. ↑ Lai, C.-C.; Liu, Y.H.; Wang, C,-Y. et al. (2020). "Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and myths". Journal of Microbiology, Immunology, and Infection 53 (3): 404–12. doi:10.1016/j.jmii.2020.02.012. PMC PMC7128959. PMID 32173241. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128959. 
  20. ↑ Rothe, C.; Schunk, M.; Sothmann, P. et al. (2020). "Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany". New England Journal of Medicine 382 (10): 970-971. doi:10.1056/NEJMc2001468. PMC PMC7120970. PMID 32003551. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120970. 
  21. ↑ Arctic Network (24 March 2020). "SARS-CoV-2". https://artic.network/ncov-2019. Retrieved 15 July 2020. 
  22. ↑ Andrews, S. (8 January 2019). "FastQC". Babraham Bioinformatics. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. 
  23. ↑ Ewals, P.; Magnusson, M.; Lundin, S. et al. (2016). "MultiQC: Summarize analysis results for multiple tools and samples in a single report". Bioinformatics 32 (19): 3047-8. doi:10.1093/bioinformatics/btw354. PMC PMC5039924. PMID 27312411. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039924. 
  24. ↑ Bolger, A.M.; Lohse, M.; Usadel, B. et al. (2014). "Trimmomatic: A flexible trimmer for Illumina sequence data". Bioinformatics 30 (15): 2114-20. doi:10.1093/bioinformatics/btu170. PMC PMC4103590. PMID 24695404. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103590. 
  25. ↑ 25.0 25.1 Li, H.; Durbin, R. (2010). "Fast and accurate long-read alignment with Burrows-Wheeler transform". Bioinformatics 26 (5): 589–95. doi:10.1093/bioinformatics/btp698. PMC PMC2828108. PMID 20080505. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2828108. 
  26. ↑ Li, H.; Handsaker, B.; Wysoker, A. et al. (2009). "The Sequence Alignment/Map format and SAMtools". Bioinformatics 25 (16): 2078-9. doi:10.1093/bioinformatics/btp352. PMC PMC2723002. PMID 19505943. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2723002. 
  27. ↑ Robinson, J.T.; Thorvaldsdóttir, H.; Winckler, W. et al. (2011). "Integrative genomics viewer". Nature Biotechnology 29 (1): 24–6. doi:10.1038/nbt.1754. PMC PMC3346182. PMID 21221095. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346182. 
  28. ↑ Narasimhan, V.; Danecek, P.; Scally, A. et al. (2016). BCFtools/RoH: A hidden Markov model approach for detecting autozygosity from next-generation sequencing data. 32. pp. 1749-51. doi:10.1093/bioinformatics/btw044. PMC PMC4892413. PMID 26826718. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892413. 
  29. ↑ "Error: no |title= specified when using {{Cite web}}". GitHub. 2020. https://github.com/lh3/seqtk. 

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.

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