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Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

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Alignments to reference sequence and hybrid reconstruction. (a) Coverage plot for 91 nanopore sequencing reads of bacteriophage phi X 174 genomic DNA. Left and right alignment bounds are indicated by the extent of the line for each read. Random attachment of the asymmetric adaptors results in reads of both sense and antisense strands. Reads below the black dashed line (events 1–38) are sense strands while reads above (events 40–92) are antisense strands. Most reads begin near the 5' end of the linearization cut site and proceed towards the 3' end as the phi29 DNAP unzips the double stranded DNA. (b) Sum total coverage for each region within the phi X 174 genome. This graph indicates the number of reads that cover any given section of the genome using the sense and antisense strands. (c) Hybrid assembly of Illumina sequencing reads using a single nanopore read (Supplementary Discussion). Thirty-eight Illumina reads (horizontal black lines) are aligned to a single 3,819 nt long nanopore read (blue trace; indicated by the red * in panel a). (d) Detail of shaded region in panel c. Six 100 bp Illumina reads are shown where they align to the nanopore read.
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Figure 4: Alignments to reference sequence and hybrid reconstruction. (a) Coverage plot for 91 nanopore sequencing reads of bacteriophage phi X 174 genomic DNA. Left and right alignment bounds are indicated by the extent of the line for each read. Random attachment of the asymmetric adaptors results in reads of both sense and antisense strands. Reads below the black dashed line (events 1–38) are sense strands while reads above (events 40–92) are antisense strands. Most reads begin near the 5' end of the linearization cut site and proceed towards the 3' end as the phi29 DNAP unzips the double stranded DNA. (b) Sum total coverage for each region within the phi X 174 genome. This graph indicates the number of reads that cover any given section of the genome using the sense and antisense strands. (c) Hybrid assembly of Illumina sequencing reads using a single nanopore read (Supplementary Discussion). Thirty-eight Illumina reads (horizontal black lines) are aligned to a single 3,819 nt long nanopore read (blue trace; indicated by the red * in panel a). (d) Detail of shaded region in panel c. Six 100 bp Illumina reads are shown where they align to the nanopore read.

Mentions: We next assessed whether we could achieve long nanopore sequencing reads. We constructed a genomic DNA sequencing library by ligating asymmetric adaptors to the linearized, full-length phi X 174 genome as described above, and this library was nanopore sequenced. We generated 106 long (>200 base-pair) ion current recordings corresponding to single molecules within this library. We aligned these reads to ion current levels predicted with the quadromer map; 92 of these reads aligned with high confidence to the phi X 174 genome (Fig. 4a) with a misalignment probability estimated at < 1×10−10. Within this set of aligned reads, ~60% were >1,000 bp, ~20% were >2,000 bp and ~10% were >3,000 bp. This is in contrast to the length distribution of our library (Supplementary Fig. 9), which contains far longer strands, implying that DNAP dissociation from the strand is the primary cause of event termination. As expected, the 5' end of most reads aligns to the cut site of the restriction enzyme used to linearize the genome, and these reads are split approximately equally between the sense and antisense strands (Fig. 4a). The 92 reads comprise a sum total of 118 kilobases (kb) with mean 21.9-fold coverage of the phi X 174 reference genome (range: 10-fold to 44-fold) (Fig. 4b).


Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

Alignments to reference sequence and hybrid reconstruction. (a) Coverage plot for 91 nanopore sequencing reads of bacteriophage phi X 174 genomic DNA. Left and right alignment bounds are indicated by the extent of the line for each read. Random attachment of the asymmetric adaptors results in reads of both sense and antisense strands. Reads below the black dashed line (events 1–38) are sense strands while reads above (events 40–92) are antisense strands. Most reads begin near the 5' end of the linearization cut site and proceed towards the 3' end as the phi29 DNAP unzips the double stranded DNA. (b) Sum total coverage for each region within the phi X 174 genome. This graph indicates the number of reads that cover any given section of the genome using the sense and antisense strands. (c) Hybrid assembly of Illumina sequencing reads using a single nanopore read (Supplementary Discussion). Thirty-eight Illumina reads (horizontal black lines) are aligned to a single 3,819 nt long nanopore read (blue trace; indicated by the red * in panel a). (d) Detail of shaded region in panel c. Six 100 bp Illumina reads are shown where they align to the nanopore read.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126851&req=5

Figure 4: Alignments to reference sequence and hybrid reconstruction. (a) Coverage plot for 91 nanopore sequencing reads of bacteriophage phi X 174 genomic DNA. Left and right alignment bounds are indicated by the extent of the line for each read. Random attachment of the asymmetric adaptors results in reads of both sense and antisense strands. Reads below the black dashed line (events 1–38) are sense strands while reads above (events 40–92) are antisense strands. Most reads begin near the 5' end of the linearization cut site and proceed towards the 3' end as the phi29 DNAP unzips the double stranded DNA. (b) Sum total coverage for each region within the phi X 174 genome. This graph indicates the number of reads that cover any given section of the genome using the sense and antisense strands. (c) Hybrid assembly of Illumina sequencing reads using a single nanopore read (Supplementary Discussion). Thirty-eight Illumina reads (horizontal black lines) are aligned to a single 3,819 nt long nanopore read (blue trace; indicated by the red * in panel a). (d) Detail of shaded region in panel c. Six 100 bp Illumina reads are shown where they align to the nanopore read.
Mentions: We next assessed whether we could achieve long nanopore sequencing reads. We constructed a genomic DNA sequencing library by ligating asymmetric adaptors to the linearized, full-length phi X 174 genome as described above, and this library was nanopore sequenced. We generated 106 long (>200 base-pair) ion current recordings corresponding to single molecules within this library. We aligned these reads to ion current levels predicted with the quadromer map; 92 of these reads aligned with high confidence to the phi X 174 genome (Fig. 4a) with a misalignment probability estimated at < 1×10−10. Within this set of aligned reads, ~60% were >1,000 bp, ~20% were >2,000 bp and ~10% were >3,000 bp. This is in contrast to the length distribution of our library (Supplementary Fig. 9), which contains far longer strands, implying that DNAP dissociation from the strand is the primary cause of event termination. As expected, the 5' end of most reads aligns to the cut site of the restriction enzyme used to linearize the genome, and these reads are split approximately equally between the sense and antisense strands (Fig. 4a). The 92 reads comprise a sum total of 118 kilobases (kb) with mean 21.9-fold coverage of the phi X 174 reference genome (range: 10-fold to 44-fold) (Fig. 4b).

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Show MeSH
Related in: MedlinePlus