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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.

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Method of construction of SMRTbell templates. (A) Method for template generation. The boxes on the left depict the process for SMRTbell generation from a PCR fragment. The boxes on the right illustrate the process for randomly generated fragments of DNA. Whereas PCR products are produced in a defined length and, if digested with a restriction enzyme, contain defined overhangs, genomic DNA must be sheared down to an appropriate size, end polished to generate blunt ends and then extended by 1 nt to generate a single A overhang. This is represented schematically with a generic ‘N’ overhang. Hairpin loops with an overhang complementary to the overhang on the DNA fragments are ligated to the ends of the insert in the final step. (B) The single-nucleotide polymorphism-containing constructs used in this work. The two templates contain an insert of ∼140 bp, with either a T/A or a G/C base pair at the site of the polymorphism (indicated in bold).
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Figure 2: Method of construction of SMRTbell templates. (A) Method for template generation. The boxes on the left depict the process for SMRTbell generation from a PCR fragment. The boxes on the right illustrate the process for randomly generated fragments of DNA. Whereas PCR products are produced in a defined length and, if digested with a restriction enzyme, contain defined overhangs, genomic DNA must be sheared down to an appropriate size, end polished to generate blunt ends and then extended by 1 nt to generate a single A overhang. This is represented schematically with a generic ‘N’ overhang. Hairpin loops with an overhang complementary to the overhang on the DNA fragments are ligated to the ends of the insert in the final step. (B) The single-nucleotide polymorphism-containing constructs used in this work. The two templates contain an insert of ∼140 bp, with either a T/A or a G/C base pair at the site of the polymorphism (indicated in bold).

Mentions: Methods for producing these structures have been described previously for short hairpin loop sequences (10–13). For templates prepared from PCR fragments, we follow a similar approach, but with modifications to the hairpin design. In this case, PCR fragments are digested with a restriction enzyme and then ligated to hairpin-forming oligonucleotides containing a complementary overhang (Figure 2A). We find that one of the by-products of ligation, dimeric and higher-order multimers of the insert, form during this ligation. These by-products are eliminated through the use of Type IIS restriction enzymes. As adapter ligation is a bimolecular process, the efficiency of ligation is relatively independent of insert size, enabling the generation of templates across a wide range of insert sizes.Figure 2.


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)

Method of construction of SMRTbell templates. (A) Method for template generation. The boxes on the left depict the process for SMRTbell generation from a PCR fragment. The boxes on the right illustrate the process for randomly generated fragments of DNA. Whereas PCR products are produced in a defined length and, if digested with a restriction enzyme, contain defined overhangs, genomic DNA must be sheared down to an appropriate size, end polished to generate blunt ends and then extended by 1 nt to generate a single A overhang. This is represented schematically with a generic ‘N’ overhang. Hairpin loops with an overhang complementary to the overhang on the DNA fragments are ligated to the ends of the insert in the final step. (B) The single-nucleotide polymorphism-containing constructs used in this work. The two templates contain an insert of ∼140 bp, with either a T/A or a G/C base pair at the site of the polymorphism (indicated in bold).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Method of construction of SMRTbell templates. (A) Method for template generation. The boxes on the left depict the process for SMRTbell generation from a PCR fragment. The boxes on the right illustrate the process for randomly generated fragments of DNA. Whereas PCR products are produced in a defined length and, if digested with a restriction enzyme, contain defined overhangs, genomic DNA must be sheared down to an appropriate size, end polished to generate blunt ends and then extended by 1 nt to generate a single A overhang. This is represented schematically with a generic ‘N’ overhang. Hairpin loops with an overhang complementary to the overhang on the DNA fragments are ligated to the ends of the insert in the final step. (B) The single-nucleotide polymorphism-containing constructs used in this work. The two templates contain an insert of ∼140 bp, with either a T/A or a G/C base pair at the site of the polymorphism (indicated in bold).
Mentions: Methods for producing these structures have been described previously for short hairpin loop sequences (10–13). For templates prepared from PCR fragments, we follow a similar approach, but with modifications to the hairpin design. In this case, PCR fragments are digested with a restriction enzyme and then ligated to hairpin-forming oligonucleotides containing a complementary overhang (Figure 2A). We find that one of the by-products of ligation, dimeric and higher-order multimers of the insert, form during this ligation. These by-products are eliminated through the use of Type IIS restriction enzymes. As adapter ligation is a bimolecular process, the efficiency of ligation is relatively independent of insert size, enabling the generation of templates across a wide range of insert sizes.Figure 2.

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.

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