Fluorogenic DNA sequencing in PDMS microreactors

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, Xin

doi:10.1038/nmeth.1629

Cited by 71 publications

(57 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 454 (1), Ion Torrent (2) and Fluorogenic sequencing (3), derived from the pyrosequencing technology (4–6), sequence DNA homopolymers by detecting by-products of the nucleotide incorporation reactions, i.e. the pyrophosphates (454), hydrogen ions (Ion Torrent) and fluorescent molecules (Fluorogenic sequencing).…”

Section: Introductionmentioning

confidence: 99%

PyroHMMsnp: an SNP caller for Ion Torrent and 454 sequencing data

Zeng

Jiang

Chen

2013

Nucleic Acids Research

View full text Add to dashboard Cite

Both 454 and Ion Torrent sequencers are capable of producing large amounts of long high-quality sequencing reads. However, as both methods sequence homopolymers in one cycle, they both suffer from homopolymer uncertainty and incorporation asynchronization. In mapping, such sequencing errors could shift alignments around homopolymers and thus induce incorrect mismatches, which have become a critical barrier against the accurate detection of single nucleotide polymorphisms (SNPs). In this article, we propose a hidden Markov model (HMM) to statistically and explicitly formulate homopolymer sequencing errors by the overcall, undercall, insertion and deletion. We use a hierarchical model to describe the sequencing and base-calling processes, and we estimate parameters of the HMM from resequencing data by an expectation-maximization algorithm. Based on the HMM, we develop a realignment-based SNP-calling program, termed PyroHMMsnp, which realigns read sequences around homopolymers according to the error model and then infers the underlying genotype by using a Bayesian approach. Simulation experiments show that the performance of PyroHMMsnp is exceptional across various sequencing coverages in terms of sensitivity, specificity and F1 measure, compared with other tools. Analysis of the human resequencing data shows that PyroHMMsnp predicts 12.9% more SNPs than Samtools while achieving a higher specificity. (http://code.google.com/p/pyrohmmsnp/).

show abstract

Section: Introductionmentioning

confidence: 99%

PyroHMMsnp: an SNP caller for Ion Torrent and 454 sequencing data

Zeng

Jiang

Chen

2013

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…lifetechnologies.com. Accessed 2013 Aug 24), and other upcoming bead-based technologies such as Fluorogenic Pyrosequencing [35]. Although the downstream readout instrumentation for the reporters could be of another nature (flow cytometer, simple flat-array or TaqMan probe-based optical readout), NGS offers unprecedented, massively parallel performance.…”

Section: Resultsmentioning

confidence: 99%

The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array

et al. 2013

View full text Add to dashboard Cite

Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.

show abstract

“…Thereby the decoding sequencing is feasible to detect the signal intensities that are proportional to the number instead of the type of incorporated nucleotides. It is the relationship between the signal intensities of effective quantitative molecules (such as pyrophosphates [3], hydrogen ions [4], or fluorescence [2,5]) and the number of incorporated nucleotides that lays theoretical foundation of this strategy.…”

Section: Proof-of-principle Experimentsmentioning

confidence: 99%

“…The existing SBS based high-throughput sequencing methodologies are classified as single nucleotide addition [2][3][4][5], and four nucleotides addition [6][7][8][9][10]. The former is to add only one kind of nucleotide into a reaction to determine the number of incorporated nucleotides; the latter is to add special modified monomers to determine the type of incorporated nucleotides.…”

Section: Introductionmentioning

confidence: 99%

A real-time decoding sequencing based on dual mononucleotide addition for cyclic synthesis

Cui

et al. 2014

Analytica Chimica Acta

View full text Add to dashboard Cite

Fluorogenic DNA sequencing in PDMS microreactors

Cited by 71 publications

References 29 publications

PyroHMMsnp: an SNP caller for Ion Torrent and 454 sequencing data

PyroHMMsnp: an SNP caller for Ion Torrent and 454 sequencing data

The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array

A real-time decoding sequencing based on dual mononucleotide addition for cyclic synthesis

Contact Info

Product

Resources

About