The removal of fluorescence in sequencing-by-synthesis

Gao, Li; Zhang, Lu

doi:10.1016/j.bbrc.2009.07.068

Cited by 6 publications

(1 citation statement)

References 24 publications

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“…SBS is a method of sequencing which utilizes a DNA Polymerase enzyme to incorporate a single fluorescently labeled nucleotide that contains a reversible terminator. This allows a period of data acquisition before removal of the fluorophore, reversal of the terminator, and continuation of sequencing [24]. Additionally, there are single molecule and real-time SBS approaches [25,26], which, as their names imply, are performed without template amplification and sequenced in real-time using some indicator of nucleotide incorporation.…”

Section: Introductionmentioning

confidence: 99%

Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides

Murphy

Wilson

et al. 2011

BMC Genomics

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BackgroundSequencing-by-ligation (SBL) is one of several next-generation sequencing methods that has been developed for massive sequencing of DNA immobilized on arrayed beads (or other clonal amplicons). SBL has the advantage of being easy to implement and accessible to all because it can be performed with off-the-shelf reagents. However, SBL has the limitation of very short read lengths.ResultsTo overcome the read length limitation, research groups have developed complex library preparation processes, which can be time-consuming, difficult, and result in low complexity libraries. Herein we describe a variation on traditional SBL protocols that extends the number of sequential bases that can be sequenced by using Endonuclease V to nick a query primer, thus leaving a ligatable end extended into the unknown sequence for further SBL cycles. To demonstrate the protocol, we constructed a known DNA sequence and utilized our SBL variation, cyclic SBL (cSBL), to resequence this region. Using our method, we were able to read thirteen contiguous bases in the 3' - 5' direction.ConclusionsCombining this read length with sequencing in the 5' - 3' direction would allow a read length of over twenty bases on a single tage. Implementing mate-paired tags and this SBL variation could enable > 95% coverage of the genome.

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Section: Introductionmentioning

confidence: 99%