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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

Bases aligned for vaccinia-Lister sample sequencing run using BLASR. The number of aligned bases for the vaccinia-Lister dataset are represented by the bars, having been aligned to a reference database containing the complete collection of known targets of the PCR primers that were used.
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Fig2: Bases aligned for vaccinia-Lister sample sequencing run using BLASR. The number of aligned bases for the vaccinia-Lister dataset are represented by the bars, having been aligned to a reference database containing the complete collection of known targets of the PCR primers that were used.

Mentions: The reference database used for this comparison was constructed from the known set of similar genes that could potentially be amplified using these primers, included in the GigaDB data [11]. The bacterial reference database included all bacterial 16S rDNA while the viral reference database included all poxvirus sequences that are potentially amplified using the consensus primer pair. During the read mapping process, all potential alignments were retained, so that there was no algorithm used to pick the most likely source of each individual read. In an attempt to represent this novel dataset in the most unbiased manner, the sum of the lengths of each alignment to a given reference was used as a simple summary statistic for the degree to which the input dataset matched that reference. Surprisingly, not only did the correct species receive the largest number of aligned bases, the correct strain within that species was also identified using that metric (Figure 2, Additional file 1: Figure S2).Figure 2


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)

Bases aligned for vaccinia-Lister sample sequencing run using BLASR. The number of aligned bases for the vaccinia-Lister dataset are represented by the bars, having been aligned to a reference database containing the complete collection of known targets of the PCR primers that were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Bases aligned for vaccinia-Lister sample sequencing run using BLASR. The number of aligned bases for the vaccinia-Lister dataset are represented by the bars, having been aligned to a reference database containing the complete collection of known targets of the PCR primers that were used.
Mentions: The reference database used for this comparison was constructed from the known set of similar genes that could potentially be amplified using these primers, included in the GigaDB data [11]. The bacterial reference database included all bacterial 16S rDNA while the viral reference database included all poxvirus sequences that are potentially amplified using the consensus primer pair. During the read mapping process, all potential alignments were retained, so that there was no algorithm used to pick the most likely source of each individual read. In an attempt to represent this novel dataset in the most unbiased manner, the sum of the lengths of each alignment to a given reference was used as a simple summary statistic for the degree to which the input dataset matched that reference. Surprisingly, not only did the correct species receive the largest number of aligned bases, the correct strain within that species was also identified using that metric (Figure 2, Additional file 1: Figure S2).Figure 2

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