Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Investigation into taxane resistant breast cancer

One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of the most significant problems with taxanes is chemoresistance. Unfortunately, some patients are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying taxane resistance. Isogenic breast cancer cell lines that were taxane resistant were generated to use as an experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of 5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included. Dose response cell count experiments were performed that confirmed taxane resistant cells had been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were maintained in a taxane free environment for six months and then rechallenged with taxane. When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored the pattern of growth of corresponding PACR cells that had been maintained in the presence of paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent phenotype. This meant that experiments could be designed to grow cell lines as xenografts in mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of taxane treatment on both native and resistant cells were analysed using flow cytometry. Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also investigated and we established that treatment with paclitaxel also exerted a G2/M block in these lines. In future studies this process will be repeated to investigate the effect of taxane treatment on the ZR75-1 PACR and DOCR lines. CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice and confirmed that all 3 groups showed very similar growth patterns. A cross resistance experiment was conducted and it was determined that the DOCR xenografts maintained a taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane resistant cell lines grown in this way have been established as xenografts in mice. These cross resistance experiments represent novel findings and merit further investigation. Extensive genomic and transcriptomic analyses were carried out on the cell lines to help identify potential taxane resistance markers. aCGH experiments were carried out to compliment the illumina experiments. The first set of experiments used DNA from pooled whole female blood as ref sample and DNA from each of the native and taxane resistant cell lines as test samples. The second set of experiments used DNA from native cells as a ref sample and DNA from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3, 11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q. For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines; RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting data was analysed to determine genes commonly dysregulated across resistance levels, between MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An extensive literature search was conducted and established four genes of interest in the context of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1. The novel biomarkers identified in the illumina experiments were validated with complimentary qPCR gene expression experiments looking at expression levels of the eleven commonly dysregulated genes identified and a panel of 19 other genes with significantly increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data from the genomic study, the gene expression profile, qPCR and Western blotting it was established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1 had increased expression in the MDA-MB-231 PACR line. Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in tumours following treatment with docetaxel and to compare them to changes identified in our in vitro and xenograft models, this allowed the final step to be taken into a translational environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in gene expression pre and post docetaxel treatment to be tracked. The gene expression data from the LAPATAX trial was combined with gene expression data from our cell line panel and identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample size is small this has provided extremely valuable evidence directly from the clinic. These two novel putative biomarkers are extremely intriguing and certainly merit further investigation, ideally using additional taxane treated breast tumour tissue. Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell lines and with two different taxanes within one cell line. The cell lines were characterised and the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines. Selected cell lines were grown as xenografts in mice and performed successful cross resistance studies upon them. A large transcriptomic and genomic analysis was conducted and has identified a panel of potential taxane resistance markers and areas of loss and gain in the genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR and Western blotting. This allowed a panel of novel taxane resistance markers to be identified. In future studies it is hoped that these targets will be knocked down with shRNA to observe if the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be conducted to find agents that may be used to reduce expression of these markers and restore sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting. As far as the author is aware this is the first time that isogenic taxane resistant cell lines have been generated and investigated in this way

Investigation into taxane resistant breast cancer

One group of chemotherapeutics that are used successfully to treat breast cancer, alone or in combination with other agents, are the taxanes; paclitaxel and docetaxel. They act by interfering with the spindle microtubule dynamics of the cell causing cell cycle arrest. However, the complexities underlying the mechanism of action are yet to be fully elucidated. Arguably, one of the most significant problems with taxanes is chemoresistance. Unfortunately, some patients are intrinsically resistant to taxanes and others acquire resistance to taxanes as treatment advances. This problem is exacerbated by a lack of understanding of the mechanisms underlying taxane resistance. Isogenic breast cancer cell lines that were taxane resistant were generated to use as an experimental model. Paclitaxel resistant (PACR) MDA-MB-231, paclitaxel resistant ZR75-1 and docetaxel resistant (DOCR) ZR75-1 cell lines were successfully generated by incrementally increasing taxane dose in respective native cell lines in vitro. An extensive characterisation of each of the resistant cell lines was conducted, focussing primarily on the 25nM resistant cells which were determined to be the most clinically relevant dose of taxane. A suboptimal dose of 5nM, a “superoptimal” dose of 50nM and the native, taxane sensitive cells was included. Dose response cell count experiments were performed that confirmed taxane resistant cells had been generated. It was shown that MDA-MB-231 native cells were more sensitive to paclitaxel than the ZR75-1 native cells, suggesting that ZR75-1 cells may already have low level inherent resistance. The MDA-MB-231 25nM PACR cells were tested to determine whether they retained PACR when maintained in media containing no paclitaxel. MDA-MB-231 25nM PACR cells were maintained in a taxane free environment for six months and then rechallenged with taxane. When rechallenged, the PACR cells previously maintained in the absence of paclitaxel mirrored the pattern of growth of corresponding PACR cells that had been maintained in the presence of paclitaxel. This proved that in the absence of paclitaxel, PACR cells did not revert to parent phenotype. This meant that experiments could be designed to grow cell lines as xenografts in mice, (in the absence of paclitaxel) & bring in vitro experiments into an in vivo setting. Effects of taxane treatment on both native and resistant cells were analysed using flow cytometry. Paclitaxel treatment exerted G2/M block in native MDA-MB-231 cells but when PACR cells were treated with the same dose of paclitaxel no G2/M block was observed, suggesting that PACR cells had developed a mechanism for escaping G2/M block. ZR75-1 native lines were also investigated and we established that treatment with paclitaxel also exerted a G2/M block in these lines. In future studies this process will be repeated to investigate the effect of taxane treatment on the ZR75-1 PACR and DOCR lines. CD 1 nude mice were injected with cells from all five cell lines to grow xenografts, unfortunately MDA-MB-231 PACR cells failed to grow so they could not be used for further xenograft experiments. PACR, DOCR and Native ZR75-1 cells did successfully grow as xenografts in mice and confirmed that all 3 groups showed very similar growth patterns. A cross resistance experiment was conducted and it was determined that the DOCR xenografts maintained a taxane resistant phenotype to docetaxel, and not paclitaxel and the PACR xenografts may be perpetuate the paclitaxel resistant phenotype in xenografts and that there may be cross resistance to docetaxel in the paclitaxel resistant xenografts. This is the first time that taxane resistant cell lines grown in this way have been established as xenografts in mice. These cross resistance experiments represent novel findings and merit further investigation. Extensive genomic and transcriptomic analyses were carried out on the cell lines to help identify potential taxane resistance markers. aCGH experiments were carried out to compliment the illumina experiments. The first set of experiments used DNA from pooled whole female blood as ref sample and DNA from each of the native and taxane resistant cell lines as test samples. The second set of experiments used DNA from native cells as a ref sample and DNA from their respective taxane resistant cells as a test, which allowed areas of loss or gain to be tracked in the genome as resistance increased. In the MDA-MB-231 cell lines the following areas of loss extended with increasing resistance: 1p36.13-q44, 6p25.3-q12, 8p, 10p, 19q, X Chr and the following areas of gain 2p25.3-23.3, 3p24.3-q13.3, 4p16.1-q12, 5q14.3-q31.1, 8q21.13-24.3, 11q15.1-q25, centromeric 12, and centromeric 14. In the ZR75-1 PACR and DOCR cell lines the areas of loss extended with increasing resistance in the following regions: 7q, 12p and 16q. For gene expression analysis RNA was extracted from the MDA-MB-231 cell lines, labelled and hybridised them to illumina human ref 8 vs. 2 chips. Data showed a progressive increase in mRNA dysregulation as paclitaxel resistance increased. Eleven genes were dysregulated across all resistance levels in the PACR MDA-MB-231 cells when compared to the relative cell lines; RGS16, CLDN1, IL7R, P&PP1R14C, COBL, TRPV4, TSPAN8, CD33, NLRP2, P13, and PAGE5. The experiment was repeated using MDA-MB-231 PACR, ZR75-1 PACR and DOCR cells and resulting data was analysed to determine genes commonly dysregulated across resistance levels, between MDA-MB-231 PACR and ZR75-1 PACR and between ZR75-1 PACR and DOCR cell lines. An extensive literature search was conducted and established four genes of interest in the context of our genomic and transcriptomic experiments including AURKA, Mdr-1, Stathmin and YY1. The novel biomarkers identified in the illumina experiments were validated with complimentary qPCR gene expression experiments looking at expression levels of the eleven commonly dysregulated genes identified and a panel of 19 other genes with significantly increased or decreased expression as resistance increased including AURKA, Mdr-1, Stathmin and YY1. Western blots were performed with lysates from the cell lines using a standard panel of predictive breast cancer markers and AURKA, Mdr-1, Stathmin and YY1. Combining the data from the genomic study, the gene expression profile, qPCR and Western blotting it was established that Mdr-1 had increased expression in the taxane resistant ZR75-1 lines and YY1 had increased expression in the MDA-MB-231 PACR line. Material from the LAPATAX trial was used to observe any transcriptomic changes occurring in tumours following treatment with docetaxel and to compare them to changes identified in our in vitro and xenograft models, this allowed the final step to be taken into a translational environment. LAPATAX (EORTC 10054) is a phase I-II study of Lapatanib and Docetaxel as neoadjuvant treatment for HER-2 +ve locally advanced/inflammatory or large operable breast cancer. Tumour material from eighteen core biopsies pre and post treatment was obtained, the mRNA was extracted, labelled and hybridised to the illumina array. This allowed the changes in gene expression pre and post docetaxel treatment to be tracked. The gene expression data from the LAPATAX trial was combined with gene expression data from our cell line panel and identified two novel putative markers of taxane resistance DUSP1 and FOS. Although sample size is small this has provided extremely valuable evidence directly from the clinic. These two novel putative biomarkers are extremely intriguing and certainly merit further investigation, ideally using additional taxane treated breast tumour tissue. Ultimately, an isogenic in vitro model of taxane resistance was developed in two different cell lines and with two different taxanes within one cell line. The cell lines were characterised and the effect of the taxanes on the cell cycle was determined in the native and taxane resistant lines. Selected cell lines were grown as xenografts in mice and performed successful cross resistance studies upon them. A large transcriptomic and genomic analysis was conducted and has identified a panel of potential taxane resistance markers and areas of loss and gain in the genome perpetuated by increasing taxane resistance. This analysis was validated using qPCR and Western blotting. This allowed a panel of novel taxane resistance markers to be identified. In future studies it is hoped that these targets will be knocked down with shRNA to observe if the taxane resistant cell lines revert to the parental phenotype. In vitro studies will be conducted to find agents that may be used to reduce expression of these markers and restore sensitivity to taxanes and consequently restore the efficacy of these drugs in a clinical setting. As far as the author is aware this is the first time that isogenic taxane resistant cell lines have been generated and investigated in this way.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Detection of norovirus by BD MAX™ Xpert ^® Norovirus, and xTAG ^® Gastrointestinal Pathogen Panel in stool and vomit samples

Background: Norovirus is a leading cause of infectious gastroenteritis, characterized by outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests allow rapid and sensitive laboratory diagnosis of norovirus, with a number of commercial platforms now available. Objectives: Evaluate the performance of the Becton Dickinson BD-MAX™System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and BD-MAX™ in vomit samples. Study design: 163 diarrhoeal stool samples were tested on four diagnostic systems (laboratory-defined real time RT-PCR (assigned as gold standard), BD MAX™ Xpert® Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. Results: In stool, sensitivity and specificity of the BD-MAX™ was 96.8% and 100%, for Xpert® Norovirus Assay was 91.9% and 100%, and for xTAG® GPP was 79.0% and 87.1%. In vomit samples positive and negative percent agreement was 95.6% and 92.0%, between the BD-MAX™ and Xpert® Norovirus. Conclusions: The BD-MAX™ System with user defined settings and the Xpert® Norovirus Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.</p

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.Publisher PDFPeer reviewe

Detection of Norovirus by BD MAX™, Xpert ® Norovirus, and xTAG ® Gastrointestinal Pathogen Panel in stool and vomit samples

Background: Norovirus is a leading cause of infectious gastroenteritis, characterized by outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests allow rapid and sensitive laboratory diagnosis of norovirus, with a number of commercial platforms now available. Objectives: Evaluate the performance of the Becton Dickinson BD-MAX™System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and BD-MAX™ in vomit samples. Study design: 163 diarrhoeal stool samples were tested on four diagnostic systems (laboratory-defined real time RT-PCR (assigned as gold standard), BD MAX™ Xpert® Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. Results: In stool, sensitivity and specificity of the BD-MAX™ was 96.8% and 100%, for Xpert® Norovirus Assay was 91.9% and 100%, and for xTAG® GPP was 79.0% and 87.1%. In vomit samples positive and negative percent agreement was 95.6% and 92.0%, between the BD-MAX™ and Xpert® Norovirus. Conclusions: The BD-MAX™ System with user defined settings and the Xpert® Norovirus Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.</p

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response

Percent genome coverage at 10× for clinical specimens sequenced with the amplicon-based sequencing approach.

(A) Clinical specimens (n = 123) consisting of 115 lesion swabs, 5 oropharyngeal swabs in the absence of lesions, and 3 oropharyngeal swabs in the presence of lesions sequenced by the MASPHL. Libraries were prepared using the Illumina DNA prep kit and sequenced on the MiSeq with 0.5–1 million reads per sample. A negative template control was included during library prep for each sequencing run. (B) Lesion swabs (n = 22) obtained from 12 individuals through the CDPH and sequenced by the YSPH. Libraries were prepared using the Illumina COVIDSeq test (RUO version) and sequenced on the NovaSeq with on average 12 million reads per sample. A negative template control was included during library prep. Bioinformatic analyses were unified between both laboratories using iVar with a minimum read depth of 10. Source data can be found in S2 Data. CDPH, Connecticut Department of Public Health; Ct, cycle threshold; MASPHL, Massachusetts State Public Health Laboratory; YSPH, Yale School of Public Health.</p

Source data for S1 Fig.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.</div