ArticleAnalytical biochemistry2007
Analysis of read length limiting factors in Pyrosequencing chemistry.
Article in Analytical biochemistry, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Methylenetetrahydrofolate Reductase CpG Islands: Epigenotyping.Journal of clinical laboratory analysis · 2016Article
- [Quality evaluation and clinical applicability of pyrosequencing assay kit for detecting hepatitis B virus resistance].Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology · 2015Article
- Isonitrosoacetophenone drives transcriptional reprogramming in Nicotiana tabacum cells in support of innate immunity and defense.PloS one · 2015Article
- Emerging technologies for the clinical microbiology laboratory.Clinical microbiology reviews · 2014Review
- Correction of sequence-dependent ambiguous bases (Ns) from the 454 pyrosequencing system.Nucleic acids research · 2014Article
- Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing.Epigenetics & chromatin · 2013Article
- Development of single-nucleotide polymorphism-based phylum-specific PCR amplification technique: application to the community analysis using ciliates as a reference organism.Molecules and cells · 2012Article
- What Next? The Next Transit from Biology to Diagnostics: Next Generation Sequencing for Immunogenetics.Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie · 2011Article
- A multi-site study using high-resolution HLA genotyping by next generation sequencing.Tissue antigens · 2011Article
- Article
- Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing.BMC research notes · 2009Article
- Metagenomic pyrosequencing and microbial identification.Clinical chemistry · 2009Review
- High-throughput homogeneous mass cleave assay technology for the diagnosis of autosomal recessive Parkinson's disease.The Journal of molecular diagnostics : JMD · 2008Article
- Simple tools for assembling and searching high-density picolitre pyrophosphate sequence data.Source code for biology and medicine · 2008Article
- The long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing.PloS one · 2008Article
- DNA pyrosequencing-based bacterial pathogen identification in a pediatric hospital setting.Journal of clinical microbiology · 2007Article
Corrections and comments
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Authors and funding
2 authors.
Funding
Abstract
Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. In this study, we have characterized the main read length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read length of both systems based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized extension due mainly to a decrease in apyrase's efficiency in degrading excess nucleotides proves to be the main limiting factor of read length. Replacing apyrase with a washing step for removal of excess nucleotide proves to be essential in improving the read length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read length of Pyrosequencing would be well beyond 300 bases.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.