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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

Overview of the FSC996 reference genome and the MinION data.The outermost track represents the reference genome with genomic positions (in kb). The blue lines provide a visual representation of every MinION read longer than 2 kb used in the analysis and its genomic alignment position (tile layers limited to 100). The black lines show the contigs generated from the assembly of Illumina Hiseq reads and their alignment positions. The inner circle shows a heat map of the GC content of the reference genome, with the brightest colour representing 20% GC and the darkest 40%.
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f2: Overview of the FSC996 reference genome and the MinION data.The outermost track represents the reference genome with genomic positions (in kb). The blue lines provide a visual representation of every MinION read longer than 2 kb used in the analysis and its genomic alignment position (tile layers limited to 100). The black lines show the contigs generated from the assembly of Illumina Hiseq reads and their alignment positions. The inner circle shows a heat map of the GC content of the reference genome, with the brightest colour representing 20% GC and the darkest 40%.

Mentions: To assess the potential effects of variation in the GC content on error rates we compared the error rates for reads in the triplicated region of the FSC996 genome, which has a relatively high GC-content as shown in Fig. 2, to those for the rest of the genome. The GC content was found to have minor effects on the error rates of reads (Fig. 1C). To see if simple sequence repeats had any effects on error rates, we mapped all 1- to 10-nucleotide long sequences present in at least five copies in the FSC996 genome. Most of these repeats consisted of monomers (n = 16269) with lengths of 5–10 bp. We also found 30 di-nucleotide repeats (mostly AT repeats) and four nine bp repeats at least five units long. The frequency of deletion errors in monomers of A or T was more than twice that for the genome as a whole (the error frequencies for A and T monomers were very similar for all error types, so they were analysed as a single group) whereas the frequency of insertion errors in A/T monomers was around half that for the whole genome (Fig. 1C). Monomers of G and C also exhibited very similar error frequencies for all error types. Like A/T monomers, G/C monomers exhibited a somewhat higher frequency of deletions and a lower frequency of insertions than the genome as a whole, but these differences were less pronounced than those observed for A/T monomers. Conversely, whereas the frequency of mismatch errors among A/T monomers was virtually identical to the value for the whole genome, that for G/C monomers was substantially higher. Repeat units of two and nine bp appeared to have higher frequencies of deletion errors than other repeat types, although this conclusion is based on relatively few observations (Supplementary Figure S5). Overall, we conclude that while the R7.3 version of the MinION system produces lower error frequencies (by around 15–40%) than the R7 system, its error rates remain substantially higher than those achieved with the PacBio system. It should be noted that this conclusion has not been verified in genomes with high GC contents; the FSC996 genome has a relatively low GC-content of 32%.


Scaffolding of a bacterial genome using MinION nanopore sequencing.

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

Overview of the FSC996 reference genome and the MinION data.The outermost track represents the reference genome with genomic positions (in kb). The blue lines provide a visual representation of every MinION read longer than 2 kb used in the analysis and its genomic alignment position (tile layers limited to 100). The black lines show the contigs generated from the assembly of Illumina Hiseq reads and their alignment positions. The inner circle shows a heat map of the GC content of the reference genome, with the brightest colour representing 20% GC and the darkest 40%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Overview of the FSC996 reference genome and the MinION data.The outermost track represents the reference genome with genomic positions (in kb). The blue lines provide a visual representation of every MinION read longer than 2 kb used in the analysis and its genomic alignment position (tile layers limited to 100). The black lines show the contigs generated from the assembly of Illumina Hiseq reads and their alignment positions. The inner circle shows a heat map of the GC content of the reference genome, with the brightest colour representing 20% GC and the darkest 40%.
Mentions: To assess the potential effects of variation in the GC content on error rates we compared the error rates for reads in the triplicated region of the FSC996 genome, which has a relatively high GC-content as shown in Fig. 2, to those for the rest of the genome. The GC content was found to have minor effects on the error rates of reads (Fig. 1C). To see if simple sequence repeats had any effects on error rates, we mapped all 1- to 10-nucleotide long sequences present in at least five copies in the FSC996 genome. Most of these repeats consisted of monomers (n = 16269) with lengths of 5–10 bp. We also found 30 di-nucleotide repeats (mostly AT repeats) and four nine bp repeats at least five units long. The frequency of deletion errors in monomers of A or T was more than twice that for the genome as a whole (the error frequencies for A and T monomers were very similar for all error types, so they were analysed as a single group) whereas the frequency of insertion errors in A/T monomers was around half that for the whole genome (Fig. 1C). Monomers of G and C also exhibited very similar error frequencies for all error types. Like A/T monomers, G/C monomers exhibited a somewhat higher frequency of deletions and a lower frequency of insertions than the genome as a whole, but these differences were less pronounced than those observed for A/T monomers. Conversely, whereas the frequency of mismatch errors among A/T monomers was virtually identical to the value for the whole genome, that for G/C monomers was substantially higher. Repeat units of two and nine bp appeared to have higher frequencies of deletion errors than other repeat types, although this conclusion is based on relatively few observations (Supplementary Figure S5). Overall, we conclude that while the R7.3 version of the MinION system produces lower error frequencies (by around 15–40%) than the R7 system, its error rates remain substantially higher than those achieved with the PacBio system. It should be noted that this conclusion has not been verified in genomes with high GC contents; the FSC996 genome has a relatively low GC-content of 32%.

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