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Virtual terminator nucleotides for next-generation DNA sequencing.

Bowers J, Mitchell J, Beer E, Buzby PR, Causey M, Efcavitch JW, Jarosz M, Krzymanska-Olejnik E, Kung L, Lipson D, Lowman GM, Marappan S, McInerney P, Platt A, Roy A, Siddiqi SM, Steinmann K, Thompson JF - Nat. Methods (2009)

Bottom Line: We synthesized reversible terminators with tethered inhibitors for next-generation sequencing.These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced.This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

View Article: PubMed Central - PubMed

Affiliation: Helicos BioSciences Corporation, Cambridge, Massachusetts, USA.

ABSTRACT
We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

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Virtual Terminator nucleotide base-by-base incorporation in a G5 homopolymerThe substrate used for testing homopolymer sequencing is shown along with successive cycles of addition of compound 22 in a solution phase reaction. Removal of the inhibitor-dye was accomplished by cleavage of the disulfide using TCEP, a reducing agent, followed by treatment with iodoacetamide to cap the free thiol. After each cycle, an aliquot of the reaction is run on an ABI3730 sequencing machine to achieve single base resolution of DNA. Length markers are shown in orange and the DNA being synthesized is shown in blue.
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Figure 1: Virtual Terminator nucleotide base-by-base incorporation in a G5 homopolymerThe substrate used for testing homopolymer sequencing is shown along with successive cycles of addition of compound 22 in a solution phase reaction. Removal of the inhibitor-dye was accomplished by cleavage of the disulfide using TCEP, a reducing agent, followed by treatment with iodoacetamide to cap the free thiol. After each cycle, an aliquot of the reaction is run on an ABI3730 sequencing machine to achieve single base resolution of DNA. Length markers are shown in orange and the DNA being synthesized is shown in blue.

Mentions: Critical to the utility of reversible terminators is the ability to reverse the inhibition prior to subsequent base additions. We used a template with five consecutive Cs and performed base addition cycles followed by removal of the inhibitor-dye. Five cycles of addition-cleavage on such a template resulted in an almost perfectly synchronous walk through the homopolymer (Figure 1). Thus, these analogs were highly effective reversible terminators.


Virtual terminator nucleotides for next-generation DNA sequencing.

Bowers J, Mitchell J, Beer E, Buzby PR, Causey M, Efcavitch JW, Jarosz M, Krzymanska-Olejnik E, Kung L, Lipson D, Lowman GM, Marappan S, McInerney P, Platt A, Roy A, Siddiqi SM, Steinmann K, Thompson JF - Nat. Methods (2009)

Virtual Terminator nucleotide base-by-base incorporation in a G5 homopolymerThe substrate used for testing homopolymer sequencing is shown along with successive cycles of addition of compound 22 in a solution phase reaction. Removal of the inhibitor-dye was accomplished by cleavage of the disulfide using TCEP, a reducing agent, followed by treatment with iodoacetamide to cap the free thiol. After each cycle, an aliquot of the reaction is run on an ABI3730 sequencing machine to achieve single base resolution of DNA. Length markers are shown in orange and the DNA being synthesized is shown in blue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719685&req=5

Figure 1: Virtual Terminator nucleotide base-by-base incorporation in a G5 homopolymerThe substrate used for testing homopolymer sequencing is shown along with successive cycles of addition of compound 22 in a solution phase reaction. Removal of the inhibitor-dye was accomplished by cleavage of the disulfide using TCEP, a reducing agent, followed by treatment with iodoacetamide to cap the free thiol. After each cycle, an aliquot of the reaction is run on an ABI3730 sequencing machine to achieve single base resolution of DNA. Length markers are shown in orange and the DNA being synthesized is shown in blue.
Mentions: Critical to the utility of reversible terminators is the ability to reverse the inhibition prior to subsequent base additions. We used a template with five consecutive Cs and performed base addition cycles followed by removal of the inhibitor-dye. Five cycles of addition-cleavage on such a template resulted in an almost perfectly synchronous walk through the homopolymer (Figure 1). Thus, these analogs were highly effective reversible terminators.

Bottom Line: We synthesized reversible terminators with tethered inhibitors for next-generation sequencing.These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced.This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

View Article: PubMed Central - PubMed

Affiliation: Helicos BioSciences Corporation, Cambridge, Massachusetts, USA.

ABSTRACT
We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

Show MeSH
Related in: MedlinePlus