Limits...
A flexible and efficient template format for circular consensus sequencing and SNP detection.

Travers KJ, Chin CS, Rank DR, Eid JS, Turner SW - Nucleic Acids Res. (2010)

Bottom Line: Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region.In this article, we present a universal method for constructing these templates, as well as an application of their use.We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

View Article: PubMed Central - PubMed

Affiliation: Pacific Biosciences, Menlo Park, CA 94025, USA.

ABSTRACT
A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

Show MeSH
Schematic of a SMRTbell™ template. (A) A SMRTbell template consists of a double-stranded region (the insert) flanked by two hairpin loops. The hairpin loops present a single-stranded region to which a sequencing primer can bind (orange). (B) As a strand-displacing polymerase (gray) extends a primer from one of the hairpin loops, it uses one strand as the template strand and displaces the other. When the polymerase returns to the 5′-end of the primer, it begins strand displacement of the primer and continues to synthesize DNA (moving in the direction of the blue arrow). Therefore, the length of sequence obtained from these templates is not limited by the insert length. Furthermore, the resulting sequence is derived from both sense- and anti-sense strands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2926623&req=5

Figure 1: Schematic of a SMRTbell™ template. (A) A SMRTbell template consists of a double-stranded region (the insert) flanked by two hairpin loops. The hairpin loops present a single-stranded region to which a sequencing primer can bind (orange). (B) As a strand-displacing polymerase (gray) extends a primer from one of the hairpin loops, it uses one strand as the template strand and displaces the other. When the polymerase returns to the 5′-end of the primer, it begins strand displacement of the primer and continues to synthesize DNA (moving in the direction of the blue arrow). Therefore, the length of sequence obtained from these templates is not limited by the insert length. Furthermore, the resulting sequence is derived from both sense- and anti-sense strands.

Mentions: In deciding on a format for SMRT™ sequencing, a number of factors were considered, including ability to accommodate a range of insert sizes, suitability for circular consensus sequencing, simplicity and speed of construction, uniformity of structure and compatibility with ZMW geometry (9). The SMRTbell template format meets all of these criteria. As depicted in Figure 1A, a SMRTbell template structurally resembles a linear double-stranded DNA fragment. At either end, the double strand is capped with a hairpin sequence, such that there are no free 5′- or 3′-ends. These hairpins contain a sequence complementary to a primer. When incubated in the presence of a DNA polymerase, the enzyme can bind to the primer/template complex, leading to a sequencing-productive complex. As the SMRTbell template is constructed starting from a double strand, it possesses complementary strand information. Therefore, in a circular consensus application, sequence information can be obtained from both the sense and antisense strands of a template, which have different sequence contexts (Figure 1B). It is expected that the performance of a polymerase will vary with sequence context. The ability to read both strands on a single DNA molecule therefore enables correction for sequence context-dependent variation.Figure 1.


A flexible and efficient template format for circular consensus sequencing and SNP detection.

Travers KJ, Chin CS, Rank DR, Eid JS, Turner SW - Nucleic Acids Res. (2010)

Schematic of a SMRTbell™ template. (A) A SMRTbell template consists of a double-stranded region (the insert) flanked by two hairpin loops. The hairpin loops present a single-stranded region to which a sequencing primer can bind (orange). (B) As a strand-displacing polymerase (gray) extends a primer from one of the hairpin loops, it uses one strand as the template strand and displaces the other. When the polymerase returns to the 5′-end of the primer, it begins strand displacement of the primer and continues to synthesize DNA (moving in the direction of the blue arrow). Therefore, the length of sequence obtained from these templates is not limited by the insert length. Furthermore, the resulting sequence is derived from both sense- and anti-sense strands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926623&req=5

Figure 1: Schematic of a SMRTbell™ template. (A) A SMRTbell template consists of a double-stranded region (the insert) flanked by two hairpin loops. The hairpin loops present a single-stranded region to which a sequencing primer can bind (orange). (B) As a strand-displacing polymerase (gray) extends a primer from one of the hairpin loops, it uses one strand as the template strand and displaces the other. When the polymerase returns to the 5′-end of the primer, it begins strand displacement of the primer and continues to synthesize DNA (moving in the direction of the blue arrow). Therefore, the length of sequence obtained from these templates is not limited by the insert length. Furthermore, the resulting sequence is derived from both sense- and anti-sense strands.
Mentions: In deciding on a format for SMRT™ sequencing, a number of factors were considered, including ability to accommodate a range of insert sizes, suitability for circular consensus sequencing, simplicity and speed of construction, uniformity of structure and compatibility with ZMW geometry (9). The SMRTbell template format meets all of these criteria. As depicted in Figure 1A, a SMRTbell template structurally resembles a linear double-stranded DNA fragment. At either end, the double strand is capped with a hairpin sequence, such that there are no free 5′- or 3′-ends. These hairpins contain a sequence complementary to a primer. When incubated in the presence of a DNA polymerase, the enzyme can bind to the primer/template complex, leading to a sequencing-productive complex. As the SMRTbell template is constructed starting from a double strand, it possesses complementary strand information. Therefore, in a circular consensus application, sequence information can be obtained from both the sense and antisense strands of a template, which have different sequence contexts (Figure 1B). It is expected that the performance of a polymerase will vary with sequence context. The ability to read both strands on a single DNA molecule therefore enables correction for sequence context-dependent variation.Figure 1.

Bottom Line: Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region.In this article, we present a universal method for constructing these templates, as well as an application of their use.We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

View Article: PubMed Central - PubMed

Affiliation: Pacific Biosciences, Menlo Park, CA 94025, USA.

ABSTRACT
A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

Show MeSH