Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing
ABSTRACT Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-ge...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-06-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00009-22 |
| _version_ | 1828803418437713920 |
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| author | Charles M. Thurlow Sandeep J. Joseph Lilia Ganova-Raeva Samantha S. Katz Lara Pereira Cheng Chen Alyssa Debra Kendra Vilfort Kimberly Workowski Stephanie E. Cohen Hilary Reno Yongcheng Sun Mark Burroughs Mili Sheth Kai-Hua Chi Damien Danavall Susan S. Philip Weiping Cao Ellen N. Kersh Allan Pillay |
| author_facet | Charles M. Thurlow Sandeep J. Joseph Lilia Ganova-Raeva Samantha S. Katz Lara Pereira Cheng Chen Alyssa Debra Kendra Vilfort Kimberly Workowski Stephanie E. Cohen Hilary Reno Yongcheng Sun Mark Burroughs Mili Sheth Kai-Hua Chi Damien Danavall Susan S. Philip Weiping Cao Ellen N. Kersh Allan Pillay |
| author_sort | Charles M. Thurlow |
| collection | DOAJ |
| description | ABSTRACT Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5′-C-phosphate-G-3′ (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now. IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum. |
| first_indexed | 2024-12-12T07:22:37Z |
| format | Article |
| id | doaj.art-5a5fa73031d84cdba93faa57d7923356 |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-12T07:22:37Z |
| publishDate | 2022-06-01 |
| publisher | American Society for Microbiology |
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| series | mSphere |
| spelling | doaj.art-5a5fa73031d84cdba93faa57d79233562022-12-22T00:33:15ZengAmerican Society for MicrobiologymSphere2379-50422022-06-017310.1128/msphere.00009-22Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome SequencingCharles M. Thurlow0Sandeep J. Joseph1Lilia Ganova-Raeva2Samantha S. Katz3Lara Pereira4Cheng Chen5Alyssa Debra6Kendra Vilfort7Kimberly Workowski8Stephanie E. Cohen9Hilary Reno10Yongcheng Sun11Mark Burroughs12Mili Sheth13Kai-Hua Chi14Damien Danavall15Susan S. Philip16Weiping Cao17Ellen N. Kersh18Allan Pillay19Division of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USASan Francisco Department of Public Health, San Francisco, California, USASt. Louis County Sexual Health Clinic, St. Louis, Missouri, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADepartment of Medicine, Emory University, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USADivision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USAABSTRACT Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5′-C-phosphate-G-3′ (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now. IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.https://journals.asm.org/doi/10.1128/msphere.00009-22DNA enrichmentTreponema pallidummetagenomicssyphiliswhole-genome sequencing |
| spellingShingle | Charles M. Thurlow Sandeep J. Joseph Lilia Ganova-Raeva Samantha S. Katz Lara Pereira Cheng Chen Alyssa Debra Kendra Vilfort Kimberly Workowski Stephanie E. Cohen Hilary Reno Yongcheng Sun Mark Burroughs Mili Sheth Kai-Hua Chi Damien Danavall Susan S. Philip Weiping Cao Ellen N. Kersh Allan Pillay Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing mSphere DNA enrichment Treponema pallidum metagenomics syphilis whole-genome sequencing |
| title | Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing |
| title_full | Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing |
| title_fullStr | Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing |
| title_full_unstemmed | Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing |
| title_short | Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing |
| title_sort | selective whole genome amplification as a tool to enrich specimens with low treponema pallidum genomic dna copies for whole genome sequencing |
| topic | DNA enrichment Treponema pallidum metagenomics syphilis whole-genome sequencing |
| url | https://journals.asm.org/doi/10.1128/msphere.00009-22 |
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