Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.
Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo...
Saved in:
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2020-12-01
|
| Series: | PLoS Genetics |
| Online Access: | https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1009170&type=printable |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849337226427629568 |
|---|---|
| author | Philipp Schwabl Jalil Maiguashca Sánchez Jaime A Costales Sofía Ocaña-Mayorga Maikell Segovia Hernán J Carrasco Carolina Hernández Juan David Ramírez Michael D Lewis Mario J Grijalva Martin S Llewellyn |
| author_facet | Philipp Schwabl Jalil Maiguashca Sánchez Jaime A Costales Sofía Ocaña-Mayorga Maikell Segovia Hernán J Carrasco Carolina Hernández Juan David Ramírez Michael D Lewis Mario J Grijalva Martin S Llewellyn |
| author_sort | Philipp Schwabl |
| collection | DOAJ |
| description | Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research. |
| format | Article |
| id | doaj-art-8c626f90dac74c6c836e77ad28ead76e |
| institution | Kabale University |
| issn | 1553-7390 1553-7404 |
| language | English |
| publishDate | 2020-12-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Genetics |
| spelling | doaj-art-8c626f90dac74c6c836e77ad28ead76e2025-08-20T03:44:46ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042020-12-011612e100917010.1371/journal.pgen.1009170Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity.Philipp SchwablJalil Maiguashca SánchezJaime A CostalesSofía Ocaña-MayorgaMaikell SegoviaHernán J CarrascoCarolina HernándezJuan David RamírezMichael D LewisMario J GrijalvaMartin S LlewellynAnalysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1009170&type=printable |
| spellingShingle | Philipp Schwabl Jalil Maiguashca Sánchez Jaime A Costales Sofía Ocaña-Mayorga Maikell Segovia Hernán J Carrasco Carolina Hernández Juan David Ramírez Michael D Lewis Mario J Grijalva Martin S Llewellyn Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. PLoS Genetics |
| title | Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. |
| title_full | Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. |
| title_fullStr | Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. |
| title_full_unstemmed | Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. |
| title_short | Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity. |
| title_sort | culture free genome wide locus sequence typing glst provides new perspectives on trypanosoma cruzi dispersal and infection complexity |
| url | https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1009170&type=printable |
| work_keys_str_mv | AT philippschwabl culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT jalilmaiguashcasanchez culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT jaimeacostales culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT sofiaocanamayorga culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT maikellsegovia culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT hernanjcarrasco culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT carolinahernandez culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT juandavidramirez culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT michaeldlewis culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT mariojgrijalva culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity AT martinsllewellyn culturefreegenomewidelocussequencetypingglstprovidesnewperspectivesontrypanosomacruzidispersalandinfectioncomplexity |