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Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer.

Kilianski A, Haas JL, Corriveau EJ, Liem AT, Willis KL, Kadavy DR, Rosenzweig CN, Minot SS - Gigascience (2015)

Bottom Line: The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%.While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology.This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

View Article: PubMed Central - PubMed

Affiliation: Edgewood Chemical Biological Center, 5183 Black Hawk Rd Bldg E3150 Rm 324, Aberdeen Proving Ground, MD 21010 USA.

ABSTRACT

Background: The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade.

Results: Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%.

Conclusions: While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

No MeSH data available.


Related in: MedlinePlus

Distribution of read alignments along the reference using BLASR. Each mapped read (only 40 reads are depicted here) is depicted by a blue bar. Percent identity to the reference is displayed by the tint, with lighter blue being more identical to the reference. A majority of the reads mapped here were approximately 70-75% identical to the reference. Many mapped reads span the entire length of the reference, represented on the x-axis, with no overhang.
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Fig1: Distribution of read alignments along the reference using BLASR. Each mapped read (only 40 reads are depicted here) is depicted by a blue bar. Percent identity to the reference is displayed by the tint, with lighter blue being more identical to the reference. A majority of the reads mapped here were approximately 70-75% identical to the reference. Many mapped reads span the entire length of the reference, represented on the x-axis, with no overhang.

Mentions: The distribution of read alignments using BLASR across the known reference sequence is shown in Figure 1, with a large proportion of reads completely spanning the amplicon. The majority of reads that align to the known reference sequence do so along the entirety of the read, with little sequence overhanging either end (Figure 1, Additional file 1: Figure S1A). The MinION™ reads generally represent the complete length of the amplicons that were applied to the nanopore sensor, and this is true not only in the vaccinia-Lister dataset but also in the datasets that generated fewer reads mapping to the amplicon (Table 1). The same trends were apparent using LAST to align the data (Table 1, Additional file 1: Figure S1B).Figure 1


Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer.

Kilianski A, Haas JL, Corriveau EJ, Liem AT, Willis KL, Kadavy DR, Rosenzweig CN, Minot SS - Gigascience (2015)

Distribution of read alignments along the reference using BLASR. Each mapped read (only 40 reads are depicted here) is depicted by a blue bar. Percent identity to the reference is displayed by the tint, with lighter blue being more identical to the reference. A majority of the reads mapped here were approximately 70-75% identical to the reference. Many mapped reads span the entire length of the reference, represented on the x-axis, with no overhang.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374364&req=5

Fig1: Distribution of read alignments along the reference using BLASR. Each mapped read (only 40 reads are depicted here) is depicted by a blue bar. Percent identity to the reference is displayed by the tint, with lighter blue being more identical to the reference. A majority of the reads mapped here were approximately 70-75% identical to the reference. Many mapped reads span the entire length of the reference, represented on the x-axis, with no overhang.
Mentions: The distribution of read alignments using BLASR across the known reference sequence is shown in Figure 1, with a large proportion of reads completely spanning the amplicon. The majority of reads that align to the known reference sequence do so along the entirety of the read, with little sequence overhanging either end (Figure 1, Additional file 1: Figure S1A). The MinION™ reads generally represent the complete length of the amplicons that were applied to the nanopore sensor, and this is true not only in the vaccinia-Lister dataset but also in the datasets that generated fewer reads mapping to the amplicon (Table 1). The same trends were apparent using LAST to align the data (Table 1, Additional file 1: Figure S1B).Figure 1

Bottom Line: The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%.While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology.This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

View Article: PubMed Central - PubMed

Affiliation: Edgewood Chemical Biological Center, 5183 Black Hawk Rd Bldg E3150 Rm 324, Aberdeen Proving Ground, MD 21010 USA.

ABSTRACT

Background: The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications. Recent reports have tested the ability of the MinION™ to act as a whole genome sequencer and have demonstrated that nanopore sequencing has tremendous potential utility. However, the current nanopore technology still has limitations with respect to error-rate, and this is problematic when attempting to assemble whole genomes without secondary rounds of sequencing to correct errors. In this study, we tested the ability of the MinION™ nanopore sequencer to accurately identify and differentiate bacterial and viral samples via directed sequencing of characteristic genes shared broadly across a target clade.

Results: Using a 6 hour sequencing run time, sufficient data were generated to identify an E. coli sample down to the species level from 16S rDNA amplicons. Three poxviruses (cowpox, vaccinia-MVA, and vaccinia-Lister) were identified and differentiated down to the strain level, despite over 98% identity between the vaccinia strains. The ability to differentiate strains by amplicon sequencing on the MinION™ was accomplished despite an observed per-base error rate of approximately 30%.

Conclusions: While nanopore sequencing, using the MinION™ platform from Oxford Nanopore in particular, continues to mature into a commercially available technology, practical uses are sought for the current versions of the technology. This study offers evidence of the utility of amplicon sequencing by demonstrating that the current versions of MinION™ technology can accurately identify and differentiate both viral and bacterial species present within biological samples via amplicon sequencing.

No MeSH data available.


Related in: MedlinePlus