Long-Read Pyrosequencing Using Pure 2′-Deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-Isomer

Gharizadeh, Baback; Nordström, Tommy; Ahmadian, Afshin; Ronaghi, Mostafa; Nyrén, Pål

doi:10.1006/abio.2001.5494

Cited by 81 publications

(83 citation statements)

References 17 publications

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“…Even triplet homopolymeric T, however, could account for some Pyrosequencing error; the incorporation of dATP-α-S nucleotide (2'-deoxyadenosine-5'-O'-1-thiotriphosphate) in Thomopolymeric regions results in uneven sequence signals and reduced data quality immediately downstream of such homopolyers [4]. During the development of the Pyrosequencing method, the natural dATP was replaced by dATP-α-S to increase the signalto-noise ratio [9].…”

Section: Discussionmentioning

confidence: 99%

“…However, SSB does improve sequence reads in general, and has recently been included in commercially available Pyrosequencing kits (Pyro Gold kit from Biotage). At present, the Pyrosequencing technology is limited to reads of up to 100 nucleotides [4]; however, efforts are under way to increase the read length. In contrast, the Sanger dideoxy sequencing method has average read lengths of 400-600 bases [12], and there are reports of Sanger sequencing reads of over 1000 bases [2].…”

Section: Discussionmentioning

confidence: 99%

“…Pyrosequencing technology [3] is emerging as an alternate method for sequencing short segments of DNA. Although the Pyrosequencing technique has had readlength limitations [4], it is a fast method with real-time readout that is highly suitable for sequencing short stretches of DNA. Unlike Sanger sequencing, which incurs a reading gap of roughly 20 bases from the primer, Pyrosequencing has the significant advantage that it can be used to read DNA immediately adjacent to the primer.…”

Section: Introductionmentioning

confidence: 99%

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Large‐scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing

et al. 2006

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Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PCR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25-30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Large‐scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing

et al. 2006

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“…These include SO 4 2− , oxyluciferin, inorganic phosphate (Pi), dNMPs and AMP. One way to limit the pool of Pi and dNMPs is to reduce the concentration of dNTPs used in PCR.…”

Section: Late-pcr Product Cleanupmentioning

confidence: 99%

“…The pyrosequencing technique has become more widely used during the last several years as reaction chemistry [3,4] and understanding of reaction kinetics [5,6] have improved, and as higher throughput hardware/software (www.pyrosequencing.com) and chemistries [7,8] have been developed. Published applications have included microbial genotyping [9][10][11][12] mutation detection [13], sequencing of heteroplasmic DNA [14], SNP genotyping [15,16], SNP allele frequency determination [17,18] SNP-based allelic loss mapping [19], gene copy number change [20] and CpG methylation analysis [21][22][23], among others.…”

Section: Introductionmentioning

confidence: 99%

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Salk¹,

Sanchez²,

Pierce³

et al. 2006

Analytical Biochemistry

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Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but is currently hampered by a labor intensive sample preparation process designed to isolate singlestranded DNA from double-stranded products generated by conventional PCR. Here LATE-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thus eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for SNP genotyping applications including an integrated, single cell-through-sequencing assay to detect a mutation at the globin IVS-110 site that is frequently responsible for β-Thalassemia.

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Determination of Nucleic Acid Sequences by Pyrosequencing

Elahi

Ronaghi²

2004

Molecular Analysis and Genome Discovery

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Long-Read Pyrosequencing Using Pure 2′-Deoxyadenosine-5′-O′-(1-thiotriphosphate) Sp-Isomer

Cited by 81 publications

References 17 publications

Large‐scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing

Large‐scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

Determination of Nucleic Acid Sequences by Pyrosequencing

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