Quantitative analysis of single‐nucleotide polymorphisms by pyrosequencing with di‐base addition

Pu, Dan; Pan, Rongfang; Liu, Wenbin; Xiao, Pengfeng

doi:10.1002/elps.201600430

Cited by 8 publications

(17 citation statements)

References 33 publications

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“…this technology is capable of detecting and quantifying allelic frequency to as low as ~5% [34]. We also concluded in our previous study that both read coverage and nucleotide frequency at a given position are significant for the SNV calling from NGS data and pyrosequencing confirmation [24].…”

Section: Resultssupporting

confidence: 62%

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing<strong> </strong>

Yang¹,

Mammel²,

Whitehouse³

et al. 2018

Preprint

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The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as Hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

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Section: Resultssupporting

confidence: 62%

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing<strong> </strong>

Yang¹,

Mammel²,

Whitehouse³

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Our previous study revealed that an intra-host heterogeneity existed in virus-infected cultured cells detected by NGS and was confirmed with pyrosequencing [ 24 ]. Pyrosequencing methods are well-established and have proven suitable for SNV analysis and viral SNV identification [ 32 , 33 ]; this technology is capable of detecting and quantifying allelic frequency to as low as ~5% [ 34 ]. We also concluded in our previous study that both read coverage and nucleotide frequency at a given position are significant for the SNV calling from NGS data and pyrosequencing confirmation [ 24 ].…”

Section: Resultsmentioning

confidence: 99%

“…In this study, in addition to ensuring the high depth of coverage for the SNVs, we employed both pyrosequencing and Illumina NGS for the SNV identification to combine both sequencing methods with complementary strengths. Pyrosequencing is a real-time sequencing method which is based on the detection of pyrophosphate released after each nucleotide incorporation in the new synthetic DNA strand, optimal for sequencing and analysis of short stretches of DNA, including SNPs [ 30 ], and has been intensively used for SNP detection since its emergence in 2005 [ 32 , 33 , 34 ]. Illumina NGS is one of the more recent sequencing technologies, it is massively parallel and allows millions of fragments to be sequenced in a single run, and it is currently used for wide applications including variation detection.…”

Section: Discussionmentioning

confidence: 99%

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

Yang

Mammel

Whitehouse

et al. 2018

Viruses

View full text Add to dashboard Cite

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

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“…It should be noted that biotin was attached to one of the DNA chains of the PCR fragment using the primer "bio-" (biotinylated). This was necessary for the analysis of the biotinylated DNA chain of the investigated amplificate using the pyrosequencing [35][36][37] . PCR primer sequences, taken for the present reseach [24,26,[28][29][30] Annealing temperature for the PCR is shown in Table 3.…”

Section: Methodsmentioning

confidence: 99%

MtDNA mutations linked with left ventricular hypertrophy

Sinyov¹,

Рыжкова²,

Sazonova³

et al. 2019

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Aim: In left ventricular hypertrophy (LVH), the heart muscle thickens. One third of individuals with LVH never complain of heart problems. However, such patients have a high risk of sudden death. LVH can be caused by arterial atherosclerotic lesions. The linkage of mtDNA mutations 652insG, m.5178C>A, m.3336T>C, m.14459G>A, 652delG, m.14846G>A, m.1555A>G, m.15059G>A, m.3256C>T, m.12315G>A and m.13513G>A with atherosclerosis was described earlier by our laboratory. The aim of the study was to analyze the linkage of these mtDNA mutations with LVH. Methods: DNA from white blood cells was isolated using a phenol-chloroform method. PCR-fragments of DNA contained the region of the investigated mutations. The heteroplasmy level of mtDNA mutations was analyzed using a pyrosequencing-based method developed by our laboratory. Results: We investigated two groups of individuals. One hundred and ninety-four patients with LVH. Two hundred and ten were conventionally healthy. It was found that mtDNA mutation m.5178C>A was significantly associated with LVH. Single nucleotide replacement m.1555A>G was associated with LVH at the level of significance P ≤ 0.1. At the same time m.12315G>A and m.3336T>C were significantly associated with the absence of this pathology. Single nucleotide replacement m.14459G>A was associated with the absence of LVH at the significance level P ≤ 0.1. Conclusion: MtDNA mutations m.5178C>A and m.1555A>G can be used for molecular genetic assessment of the predisposition of individuals to the occurrence of left ventricular hypertrophy. They can also be used for the family analysis of this pathology. Mutations m.12315G>A, m.3336T>C and m.14459G>A can be used in the development of LVH gene therapy methods.

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Quantitative analysis of single‐nucleotide polymorphisms by pyrosequencing with di‐base addition

Cited by 8 publications

References 33 publications

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing<strong> </strong>

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing<strong> </strong>

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing

MtDNA mutations linked with left ventricular hypertrophy

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