Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.

<h4>Aims</h4>The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS).<h4>Methods&...

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Autores principales:	Mukhlid Yousif, Trevor G Bell, Hatim Mudawi, Dieter Glebe, Anna Kramvis
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/258fed62485c4bba853f1bce41e9dc1d
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spelling	oai:doaj.org-article:258fed62485c4bba853f1bce41e9dc1d2021-11-18T08:22:51ZAnalysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.1932-620310.1371/journal.pone.0095377https://doaj.org/article/258fed62485c4bba853f1bce41e9dc1d2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24740330/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Aims</h4>The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS).<h4>Methods</h4>Four serum samples, infected with either genotype D or E, from HBeAg-positive and HBeAg-negative patients were randomly selected. UDPS and CBS were used to sequence the basic core promoter/precore region of HBV. Two online bioinformatics tools, the "Deep Threshold Tool" and the "Rosetta Tool" (http://hvdr.bioinf.wits.ac.za/tools/), were built to test and analyze the generated data.<h4>Results</h4>A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. In the four samples, substitutions, detected at 0.5% threshold or above, were identified at 39 unique positions, 25 of which were non-synonymous mutations. Sample #2 (HBeAg-negative, genotype D) had substitutions in 26 positions, followed by sample #1 (HBeAg-negative, genotype E) in 12 positions, sample #3 (HBeAg-positive, genotype D) in 7 positions and sample #4 (HBeAg-positive, genotype E) in only four positions. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5 ∶ 1. Compared to genotype E isolates, genotype D isolates showed greater variation in the X, basic core promoter/precore and core regions. Only 18 of the 39 positions identified by UDPS were detected by CBS, which detected 14 of the 25 non-synonymous mutations detected by UDPS.<h4>Conclusion</h4>UDPS data should be approached with caution. Appropriate curation of read data is required prior to analysis, in order to clean the data and eliminate artefacts. CBS detected fewer than 50% of the substitutions detected by UDPS. Furthermore it is important that the appropriate consensus (reference) sequence is used in order to identify variants correctly.Mukhlid YousifTrevor G BellHatim MudawiDieter GlebeAnna KramvisPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 4, p e95377 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mukhlid Yousif Trevor G Bell Hatim Mudawi Dieter Glebe Anna Kramvis Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
description	<h4>Aims</h4>The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS).<h4>Methods</h4>Four serum samples, infected with either genotype D or E, from HBeAg-positive and HBeAg-negative patients were randomly selected. UDPS and CBS were used to sequence the basic core promoter/precore region of HBV. Two online bioinformatics tools, the "Deep Threshold Tool" and the "Rosetta Tool" (http://hvdr.bioinf.wits.ac.za/tools/), were built to test and analyze the generated data.<h4>Results</h4>A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. In the four samples, substitutions, detected at 0.5% threshold or above, were identified at 39 unique positions, 25 of which were non-synonymous mutations. Sample #2 (HBeAg-negative, genotype D) had substitutions in 26 positions, followed by sample #1 (HBeAg-negative, genotype E) in 12 positions, sample #3 (HBeAg-positive, genotype D) in 7 positions and sample #4 (HBeAg-positive, genotype E) in only four positions. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5 ∶ 1. Compared to genotype E isolates, genotype D isolates showed greater variation in the X, basic core promoter/precore and core regions. Only 18 of the 39 positions identified by UDPS were detected by CBS, which detected 14 of the 25 non-synonymous mutations detected by UDPS.<h4>Conclusion</h4>UDPS data should be approached with caution. Appropriate curation of read data is required prior to analysis, in order to clean the data and eliminate artefacts. CBS detected fewer than 50% of the substitutions detected by UDPS. Furthermore it is important that the appropriate consensus (reference) sequence is used in order to identify variants correctly.
format	article
author	Mukhlid Yousif Trevor G Bell Hatim Mudawi Dieter Glebe Anna Kramvis
author_facet	Mukhlid Yousif Trevor G Bell Hatim Mudawi Dieter Glebe Anna Kramvis
author_sort	Mukhlid Yousif
title	Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
title_short	Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
title_full	Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
title_fullStr	Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
title_full_unstemmed	Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
title_sort	analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis b virus using newly developed bioinformatics tools.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/258fed62485c4bba853f1bce41e9dc1d
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Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.

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