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Scaffolding of a bacterial genome using MinION nanopore sequencing.

Karlsson E, Lärkeryd A, Sjödin A, Forsman M, Stenberg P - Sci Rep (2015)

Bottom Line: The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores.We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads.With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

No MeSH data available.


Related in: MedlinePlus

Quality of MinION (R7.3) and PacBio sequencing reads.(a) Length distribution of the reads. MinION reads are divided into three length categories that are coloured separately. Note that the high number of MinION reads of about 3.5 kb originate from the ligation control fragment. (b) Mapability of PacBio and MinION reads divided into the same length categories as in (a). Read alignment length is the fraction of the reads covered in the BLAST alignment against the reference genome. (c) Mean frequencies of deletion, insertion and substitution errors per nucleotide per read for MinION sequence reads in four genomic regions: whole genome (32% GC), high GC-content regions (47.8% GC), A/T and G/C monomers at least 5 bp long. (d) Consensus accuracy versus average read coverage of the genome. Different coverages were obtained by subsampling the reads from a single MinION run.
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f1: Quality of MinION (R7.3) and PacBio sequencing reads.(a) Length distribution of the reads. MinION reads are divided into three length categories that are coloured separately. Note that the high number of MinION reads of about 3.5 kb originate from the ligation control fragment. (b) Mapability of PacBio and MinION reads divided into the same length categories as in (a). Read alignment length is the fraction of the reads covered in the BLAST alignment against the reference genome. (c) Mean frequencies of deletion, insertion and substitution errors per nucleotide per read for MinION sequence reads in four genomic regions: whole genome (32% GC), high GC-content regions (47.8% GC), A/T and G/C monomers at least 5 bp long. (d) Consensus accuracy versus average read coverage of the genome. Different coverages were obtained by subsampling the reads from a single MinION run.

Mentions: The MinION run produced 61236 sequencing reads of which 19196 (31.3%) were classified as “2D pass” and 11227 (18.3%) as “2D fail” by the MetrichorTM Agent. The remainder consisted of template or complement-only reads. The mean and maximum lengths of the 2D pass reads were 6402 and 26651 base pairs, respectively (for comparisons with the results obtained using the R7 cell, see Supplementary Table S1). The complete read length distribution is shown in Fig. 1A; note that the peak at 3.5 kb originates from a 3560 bp ligation control added during library preparation. For a comparison of the results obtained using the R7.3 and R7 cells, see Supplementary Figure S1.


Scaffolding of a bacterial genome using MinION nanopore sequencing.

Karlsson E, Lärkeryd A, Sjödin A, Forsman M, Stenberg P - Sci Rep (2015)

Quality of MinION (R7.3) and PacBio sequencing reads.(a) Length distribution of the reads. MinION reads are divided into three length categories that are coloured separately. Note that the high number of MinION reads of about 3.5 kb originate from the ligation control fragment. (b) Mapability of PacBio and MinION reads divided into the same length categories as in (a). Read alignment length is the fraction of the reads covered in the BLAST alignment against the reference genome. (c) Mean frequencies of deletion, insertion and substitution errors per nucleotide per read for MinION sequence reads in four genomic regions: whole genome (32% GC), high GC-content regions (47.8% GC), A/T and G/C monomers at least 5 bp long. (d) Consensus accuracy versus average read coverage of the genome. Different coverages were obtained by subsampling the reads from a single MinION run.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493687&req=5

f1: Quality of MinION (R7.3) and PacBio sequencing reads.(a) Length distribution of the reads. MinION reads are divided into three length categories that are coloured separately. Note that the high number of MinION reads of about 3.5 kb originate from the ligation control fragment. (b) Mapability of PacBio and MinION reads divided into the same length categories as in (a). Read alignment length is the fraction of the reads covered in the BLAST alignment against the reference genome. (c) Mean frequencies of deletion, insertion and substitution errors per nucleotide per read for MinION sequence reads in four genomic regions: whole genome (32% GC), high GC-content regions (47.8% GC), A/T and G/C monomers at least 5 bp long. (d) Consensus accuracy versus average read coverage of the genome. Different coverages were obtained by subsampling the reads from a single MinION run.
Mentions: The MinION run produced 61236 sequencing reads of which 19196 (31.3%) were classified as “2D pass” and 11227 (18.3%) as “2D fail” by the MetrichorTM Agent. The remainder consisted of template or complement-only reads. The mean and maximum lengths of the 2D pass reads were 6402 and 26651 base pairs, respectively (for comparisons with the results obtained using the R7 cell, see Supplementary Table S1). The complete read length distribution is shown in Fig. 1A; note that the peak at 3.5 kb originates from a 3560 bp ligation control added during library preparation. For a comparison of the results obtained using the R7.3 and R7 cells, see Supplementary Figure S1.

Bottom Line: The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores.We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads.With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

No MeSH data available.


Related in: MedlinePlus