Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing

Thurlow, Charles M.; Joseph, Sandeep J.; Ganova-Raeva, Lilia; Katz, Samantha S.; Pereira, Lara; Chen, Cheng; Debra, Alyssa; Vilfort, Kendra; Workowski, Kimberly; Cohen, Stephanie E.; Reno, Hilary; Sun, Yongcheng; Burroughs, Mark; Sheth, Mili; Chi, Kai-Hua; Danavall, Damien; Philip, Susan S.; Cao, Weiping; Kersh, Ellen N.; Pillay, Allan

doi:10.1128/msphere.00009-22

Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing

Journal Article · Mon May 02 00:00:00 EDT 2022 · mSphere

DOI:https://doi.org/10.1128/msphere.00009-22· OSTI ID:1982985

^[1]; ^[1]; Ganova-Raeva, Lilia ^[1]; Katz, Samantha S. ^[1]; Pereira, Lara ^[1]; Chen, Cheng ^[1]; Debra, Alyssa ^[1]; Vilfort, Kendra ^[1]; Workowski, Kimberly ^[2]; Cohen, Stephanie E. ^[3]; Reno, Hilary ^[4]; Sun, Yongcheng ^[1]; Burroughs, Mark ^[1]; Sheth, Mili ^[1]; Chi, Kai-Hua ^[1]; Danavall, Damien ^[1]; Philip, Susan S. ^[5]; Cao, Weiping ^[1]; ^[1]; ^[1]

Centers for Disease Control and Prevention (CDC), Atlanta, GA (United States)
Centers for Disease Control and Prevention (CDC), Atlanta, GA (United States); Emory Univ., Atlanta, GA (United States)
San Francisco Department of Public Health, San Francisco, CA (United States)
St. Louis County Sexual Health Clinic, St. Louis, MO (United States); Washington Univ., St. Louis, MO (United States)
Emory Univ., Atlanta, GA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)

Sponsoring Organization:: Centers for Disease Control and Prevention (CDC); USDOE Office of Science (SC)

Grant/Contract Number:: SC0014664

OSTI ID:: 1982985

Journal Information:: mSphere, Journal Name: mSphere Journal Issue: 3 Vol. 7; ISSN 2379-5042

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (42)

The use of next generation sequencing in the diagnosis and typing of respiratory infections Thorburn, Fiona; Bennett, Susan; Modha, Sejal Journal of Clinical Virology, Vol. 69 https://doi.org/10.1016/j.jcv.2015.06.082	journal	August 2015
Next-Generation Sequencing for Infectious Disease Diagnosis and Management Lefterova, Martina I.; Suarez, Carlos J.; Banaei, Niaz The Journal of Molecular Diagnostics, Vol. 17, Issue 6 https://doi.org/10.1016/j.jmoldx.2015.07.004	journal	November 2015
Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing Thoendel, Matthew; Jeraldo, Patricio R.; Greenwood-Quaintance, Kerryl E. Journal of Microbiological Methods, Vol. 127 https://doi.org/10.1016/j.mimet.2016.05.022	journal	August 2016
Genomes of cryptic chimpanzee Plasmodium species reveal key evolutionary events leading to human malaria Sundararaman, Sesh A.; Plenderleith, Lindsey J.; Liu, Weimin Nature Communications, Vol. 7, Issue 1 https://doi.org/10.1038/ncomms11078	journal	March 2016
Fast gapped-read alignment with Bowtie 2 Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 9, Issue 4 https://doi.org/10.1038/nmeth.1923	journal	March 2012
MetaPhlAn2 for enhanced metagenomic taxonomic profiling Truong, Duy Tin; Franzosa, Eric A.; Tickle, Timothy L. Nature Methods, Vol. 12, Issue 10 https://doi.org/10.1038/nmeth.3589	journal	September 2015
Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation Pinto, Miguel; Borges, Vítor; Antelo, Minia Nature Microbiology, Vol. 2, Issue 1 https://doi.org/10.1038/nmicrobiol.2016.190	journal	October 2016
Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster Arora, Natasha; Schuenemann, Verena J.; Jäger, Günter Nature Microbiology, Vol. 2, Issue 1 https://doi.org/10.1038/nmicrobiol.2016.245	journal	December 2016
Genomic epidemiology of syphilis reveals independent emergence of macrolide resistance across multiple circulating lineages Beale, Mathew A.; Marks, Michael; Sahi, Sharon K. Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-11216-7	journal	July 2019
Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis Beale, Mathew A.; Marks, Michael; Cole, Michelle J. Nature Microbiology, Vol. 6, Issue 12 https://doi.org/10.1038/s41564-021-01000-z	journal	November 2021
Macrolide Resistance inTreponema pallidumin the United States and Ireland Lukehart, Sheila A.; Godornes, Charmie; Molini, Barbara J. New England Journal of Medicine, Vol. 351, Issue 2 https://doi.org/10.1056/NEJMoa040216	journal	July 2004
Enhanced Molecular Typing ofTreponema pallidum: Geographical Distribution of Strain Types and Association with Neurosyphilis Marra, Christina M.; Sahi, Sharon K.; Tantalo, Lauren C. The Journal of Infectious Diseases, Vol. 202, Issue 9 https://doi.org/10.1086/656533	journal	November 2010
SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry Journal of Computational Biology, Vol. 19, Issue 5 https://doi.org/10.1089/cmb.2012.0021	journal	May 2012
ABACAS: algorithm-based automatic contiguation of assembled sequences Assefa, S.; Keane, T. M.; Otto, T. D. Bioinformatics, Vol. 25, Issue 15 https://doi.org/10.1093/bioinformatics/btp347	journal	June 2009
The Sequence Alignment/Map format and SAMtools Li, H.; Handsaker, B.; Wysoker, A. Bioinformatics, Vol. 25, Issue 16 https://doi.org/10.1093/bioinformatics/btp352	journal	June 2009
BEDTools: a flexible suite of utilities for comparing genomic features Quinlan, Aaron R.; Hall, Ira M. Bioinformatics, Vol. 26, Issue 6 https://doi.org/10.1093/bioinformatics/btq033	journal	January 2010
RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies Stamatakis, Alexandros Bioinformatics, Vol. 30, Issue 9 https://doi.org/10.1093/bioinformatics/btu033	journal	January 2014
swga: a primer design toolkit for selective whole genome amplification Clarke, Erik L.; Sundararaman, Sesh A.; Seifert, Stephanie N. Bioinformatics, Vol. 33, Issue 14 https://doi.org/10.1093/bioinformatics/btx118	journal	February 2017
Analysis of Treponema pallidum Strains From China Using Improved Methods for Whole-Genome Sequencing From Primary Syphilis Chancres Chen, Wentao; Šmajs, David; Hu, Yongfei The Journal of Infectious Diseases, Vol. 223, Issue 5 https://doi.org/10.1093/infdis/jiaa449	journal	July 2020
Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins Croucher, Nicholas J.; Page, Andrew J.; Connor, Thomas R. Nucleic Acids Research, Vol. 43, Issue 3 https://doi.org/10.1093/nar/gku1196	journal	November 2014
k-SLAM: accurate and ultra-fast taxonomic classification and gene identification for large metagenomic data sets Ainsworth, David; Sternberg, Michael J. E.; Raczy, Come Nucleic Acids Research https://doi.org/10.1093/nar/gkw1248	journal	December 2016
In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique Povilaitis, Tadas; Alzbutas, Gediminas; Sukackaite, Rasa Protein Engineering, Design and Selection, Vol. 29, Issue 12 https://doi.org/10.1093/protein/gzw052	journal	November 2016
Increased Discrimination of Treponema pallidum Strains by Subtyping With a 4-Component System Incorporating a Mononucleotide Tandem Repeat in rpsA Pillay, Allan; Lee, Min-Kuang; Slezak, Tom Sexually Transmitted Diseases, Vol. 46, Issue 4 https://doi.org/10.1097/OLQ.0000000000000935	journal	April 2019
Molecular Epidemiology of Syphilis—San Francisco, 2004-2007 Katz, Kenneth A.; Pillay, Allan; Ahrens, Katherine Sexually Transmitted Diseases, Vol. 37, Issue 10 https://doi.org/10.1097/OLQ.0b013e3181e1a77a	journal	October 2010
Syphilis testing, typing, and treatment follow-up Tipple, Craig; Taylor, Graham P. Current Opinion in Infectious Diseases, Vol. 28, Issue 1 https://doi.org/10.1097/QCO.0000000000000124	journal	February 2015
Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation Itsko, Mark; Retchless, Adam C.; Joseph, Sandeep J. Journal of Clinical Microbiology, Vol. 58, Issue 12 https://doi.org/10.1128/JCM.01780-20	journal	November 2020
Detection of the A2058G and A2059G 23S rRNA Gene Point Mutations Associated with Azithromycin Resistance in Treponema pallidum by Use of a TaqMan Real-Time Multiplex PCR Assay Chen, Cheng-Yen; Chi, Kai-Hua; Pillay, Allan Journal of Clinical Microbiology, Vol. 51, Issue 3 https://doi.org/10.1128/JCM.02770-12	journal	March 2013
Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum Edmondson, Diane G.; Hu, Bo; Norris, Steven J. mBio, Vol. 9, Issue 3 https://doi.org/10.1128/mBio.01153-18	journal	June 2018
Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples Cowell, Annie N.; Loy, Dorothy E.; Sundararaman, Sesh A. mBio, Vol. 8, Issue 1 https://doi.org/10.1128/mBio.02257-16	journal	March 2017
Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains Edmondson, Diane G.; DeLay, Bridget D.; Kowis, Lindsay E. mBio, Vol. 12, Issue 1 https://doi.org/10.1128/mBio.03536-20	journal	February 2021
Whole Genome Sequences of Three Treponema pallidum ssp. pertenue Strains: Yaws and Syphilis Treponemes Differ in Less than 0.2% of the Genome Sequence Čejková, Darina; Zobaníková, Marie; Chen, Lei PLoS Neglected Tropical Diseases, Vol. 6, Issue 1 https://doi.org/10.1371/journal.pntd.0001471	journal	January 2012
Resequencing of Treponema pallidum ssp. pallidum Strains Nichols and SS14: Correction of Sequencing Errors Resulted in Increased Separation of Syphilis Treponeme Subclusters Pětrošová, Helena; Pospíšilová, Petra; Strouhal, Michal PLoS ONE, Vol. 8, Issue 9 https://doi.org/10.1371/journal.pone.0074319	journal	September 2013
A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA Feehery, George R.; Yigit, Erbay; Oyola, Samuel O. PLoS ONE, Vol. 8, Issue 10 https://doi.org/10.1371/journal.pone.0076096	journal	October 2013
Molecular characterization of Treponema pallidum subsp. pallidum in Switzerland and France with a new multilocus sequence typing scheme Grillová, Linda; Bawa, Tanika; Mikalová, Lenka PLOS ONE, Vol. 13, Issue 7 https://doi.org/10.1371/journal.pone.0200773	journal	July 2018
Successful isolation of Treponema pallidum strains from patients’ cryopreserved ulcer exudate using the rabbit model Pereira, Lara E.; Katz, Samantha S.; Sun, Yongcheng PLOS ONE, Vol. 15, Issue 1 https://doi.org/10.1371/journal.pone.0227769	journal	January 2020
Cutadapt removes adapter sequences from high-throughput sequencing reads Martin, Marcel EMBnet.journal, Vol. 17, Issue 1 https://doi.org/10.14806/ej.17.1.200	journal	May 2011
Selective Whole Genome Amplification for Resequencing Target Microbial Species from Complex Natural Samples Leichty, Aaron R.; Brisson, Dustin Genetics, Vol. 198, Issue 2 https://doi.org/10.1534/genetics.114.165498	journal	August 2014
Tracing the origin of Treponema pallidum in China using next-generation sequencing Sun, Jun; Meng, Zhefeng; Wu, Kaiqi Oncotarget, Vol. 7, Issue 28 https://doi.org/10.18632/oncotarget.10154	journal	June 2016
Directly Sequenced Genomes of Contemporary Strains of Syphilis Reveal Recombination-Driven Diversity in Genes Encoding Predicted Surface-Exposed Antigens Grillová, Linda; Oppelt, Jan; Mikalová, Lenka Frontiers in Microbiology, Vol. 10 https://doi.org/10.3389/fmicb.2019.01691	journal	July 2019
Advances in Clinical Sample Preparation for Identification and Characterization of Bacterial Pathogens Using Metagenomics Bachmann, Nathan L.; Rockett, Rebecca J.; Timms, Verlaine Joy Frontiers in Public Health, Vol. 6 https://doi.org/10.3389/fpubh.2018.00363	journal	December 2018
Molecular Differentiation of Treponema pallidum Subspecies in Skin Ulceration Clinically Suspected as Yaws in Vanuatu Using Real-Time Multiplex PCR and Serological Methods Chi, Kai-Hua; Kool, Jacob L.; Asiedu, Kingsley The American Journal of Tropical Medicine and Hygiene, Vol. 92, Issue 1 https://doi.org/10.4269/ajtmh.14-0459	journal	January 2015
Macrolide Resistance in the Syphilis Spirochete, Treponema pallidum ssp. pallidum: Can We Also Expect Macrolide-Resistant Yaws Strains? Šmajs, David; Grillová, Linda; Paštěková, Lenka The American Journal of Tropical Medicine and Hygiene, Vol. 93, Issue 4 https://doi.org/10.4269/ajtmh.15-0316	journal	October 2015

Similar Records

Complete genome sequence of Treponema pallidum strain DAL-1

Journal Article · Tue Oct 09 20:00:00 EDT 2012 · Standards in Genomic Sciences · OSTI ID:1628654

Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

Journal Article · Tue May 03 20:00:00 EDT 2016 · PLoS ONE · OSTI ID:1266216

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA environment
Metagenomics
Microbiology
Syphilis
Treponema pallidum
Whole-genome sequencing

Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing

Citation Formats

References (42)

Similar Records

Related Subjects