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Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis

To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one cult...

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Detalles Bibliográficos
Autores principales: Mishra, Deepanshi, Satpathy, Gita, Chawla, Rohan, Paliwal, Daizy, Panda, Subrat Kumar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099753/
https://www.ncbi.nlm.nih.gov/pubmed/33997374
http://dx.doi.org/10.1016/j.heliyon.2021.e06780
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author Mishra, Deepanshi
Satpathy, Gita
Chawla, Rohan
Paliwal, Daizy
Panda, Subrat Kumar
author_facet Mishra, Deepanshi
Satpathy, Gita
Chawla, Rohan
Paliwal, Daizy
Panda, Subrat Kumar
author_sort Mishra, Deepanshi
collection PubMed
description To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one culture positive endophthalmitis patients, admitted to a tertiary care hospital were processed for targeted metagenomics. NGS of 7 variable regions of 16S rRNA gene was done using Ion Torrent Personal Genome Machine (PGM). Sequence data were analyzed using Ion Reporter software using QIIME and BLSATN tools and Greengenes and NCBI–Genbank databases. Bacterial genome sequences were detected in 15 culture negative and culture positive vitreous specimens. The sequence reads varied between 25,245–540,916 with read length between 142bp–228bp and coverage depth was 41.0X and 81.2X. Operational taxonomic unit (OTUs) of multiple bacterial genera and species were detected in 13 culture negative vitreous specimens and OTUs of a single bacterial species were detected in 2 culture negative and 1 culture positive specimens; one negative specimen had no bacterial DNA. Maximum numbers of OTUs detected by NGS for a bacterial species from any vitreous specimen was the one which was detected and identified by Sanger's sequencing in broad range PCR. All the bacteria were belonging to clinically relevant species. Broad range PCR with sequencing failed to identify bacteria from 5 of the 16 (31.25%) culture negative vitreous specimens. Metagenomics could detect and identify bacterial pathogens in 15 of the 16 culture negative vitreous specimen's up to species level. With rapidly decreasing cost, metagenomics has a potential to be used widely in endophthalmitis diagnosis, in which culture negativity is usually high.
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spelling pubmed-80997532021-05-13 Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis Mishra, Deepanshi Satpathy, Gita Chawla, Rohan Paliwal, Daizy Panda, Subrat Kumar Heliyon Research Article To study the feasibility of 16S rRNA metagenomics using next generation sequencing (NGS) along with broad range PCR assay for 762 bp region of 16S rRNA gene with Sanger's sequencing, in microbial diagnosis of culture negative endophthalmitis. Vitreous fluid from 16 culture negative and one culture positive endophthalmitis patients, admitted to a tertiary care hospital were processed for targeted metagenomics. NGS of 7 variable regions of 16S rRNA gene was done using Ion Torrent Personal Genome Machine (PGM). Sequence data were analyzed using Ion Reporter software using QIIME and BLSATN tools and Greengenes and NCBI–Genbank databases. Bacterial genome sequences were detected in 15 culture negative and culture positive vitreous specimens. The sequence reads varied between 25,245–540,916 with read length between 142bp–228bp and coverage depth was 41.0X and 81.2X. Operational taxonomic unit (OTUs) of multiple bacterial genera and species were detected in 13 culture negative vitreous specimens and OTUs of a single bacterial species were detected in 2 culture negative and 1 culture positive specimens; one negative specimen had no bacterial DNA. Maximum numbers of OTUs detected by NGS for a bacterial species from any vitreous specimen was the one which was detected and identified by Sanger's sequencing in broad range PCR. All the bacteria were belonging to clinically relevant species. Broad range PCR with sequencing failed to identify bacteria from 5 of the 16 (31.25%) culture negative vitreous specimens. Metagenomics could detect and identify bacterial pathogens in 15 of the 16 culture negative vitreous specimen's up to species level. With rapidly decreasing cost, metagenomics has a potential to be used widely in endophthalmitis diagnosis, in which culture negativity is usually high. Elsevier 2021-04-23 /pmc/articles/PMC8099753/ /pubmed/33997374 http://dx.doi.org/10.1016/j.heliyon.2021.e06780 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Mishra, Deepanshi
Satpathy, Gita
Chawla, Rohan
Paliwal, Daizy
Panda, Subrat Kumar
Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title_full Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title_fullStr Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title_full_unstemmed Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title_short Targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
title_sort targeted metagenomics using next generation sequencing in laboratory diagnosis of culture negative endophthalmitis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099753/
https://www.ncbi.nlm.nih.gov/pubmed/33997374
http://dx.doi.org/10.1016/j.heliyon.2021.e06780
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