Nanopore-based DNA sequencing in clinical microbiology: preliminary assessment of basic requirements
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Abstract
Aim
Identify basic requirements for a metagenomic nanopore sequencing protocol permitting frequent application in a clinical microbiology daily routine diagnostic setting.
Background
Nanopore sequencing with the Oxford Nanopore Technologies MinION device has a potential to markedly improve clinical diagnosis of infections. Reports have emerged recently that it may provide direct-from-clinical-sample information; for example, with urine samples, bronchial tuberculosis samples and orthopedic prostheses. However, the ideal protocol for clinical use remains to be determined, especially relating to detection of relevant pathogen quantities and to finding a reasonable level of economic costs.
Results
MinION can provide qualitatively and quantitatively correct identification of multiple species in metagenomics samples. For detection of clinically relevant quantities of bacteria (on a nanogram DNA level) there is a need for carrier DNA. Importantly, high-purity DNA and a naïve MinION flow cell seem to be critical parameters.
Conclusions
Our results suggest that high-purity clinical sample DNA, addition of carrier DNA and a naïve flow cell are critical factors for clinical use of MinION. A relatively high error rate may limit detection of antimicrobial resistance genes, and a realistic level of costs will require availability of a price-reduced and single-use flowcell.
Article activity feed
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MinION promises to become able to function as a rapid and accurate pathogen species identification technique for clinical specimens.
It could be nice to comment here on how you think this might compare to the use of Illumina sequencing for clinical detection. Short read sequencing usually requires lower DNA input and sequencing can be highly multiplexed. The ability to do MinION in-house and get immediate results is definitely appealing- but how does this balance out with DNA requirements, accuracy of detection, etc.? Could be interesting to discuss briefly here.
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Figure 1)
Can you explain in the legend for this figure what is meant my the anticipated number of reads? Is this a % of the total read output based on input DNA %? I think that '4 hr MinION run' may not be the most informative title for this figure. You could consider removing the blue background and the text/title at the top to make this figure look cleaner. I also do not think it is necessary to include the ng information here. Species names could be italicized.
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Antimicrobial resistance application (AMRA)
It would be nice to provide a reference here and maybe a sentence or two on why you selected this application, since there are a number of applications that find AMR genes in sequence data. Is the detection performed on raw reads and do you think that detection may be more accurate in assembled data?
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informative reads
can you clarify what you mean by an 'informative' read?
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(Table 1)
it may be nice to include the genome coverage information in this table, or include the genome size so that the reader can estimate coverage
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customized MinKNOW
Can you be more specific here about what customized scripts were used/ provide a reference or link?
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The MINION sequencing was performed with R9.4 flow cells
Was an entire minion flow cell used for each sample that was sequenced? I see in table 1 that the run time was very short, suggesting that a full cell was not used. Either way it would be nice to explicitly state this here so that the reader can better evaluate your sequencing results. If any flow cells were reused for any experiments, it would be good to add this information to the methods, including the relevant reagent information if washes were done.
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Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF MS microflex LT from Bruker Daltonics, Bremen, Germany) was used, with scores >2 verifying the strain identity before analysis.
I think this section may benefit from a more detailed description of the method. If this is a very commonly used protocol, it would be nice to provide a reference to the protocol.
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‥… it will make a tremendous difference both to diagnostics and to science it will quickly make its way into many labs and the clinic’
I think your punctuation might be off here. It looks like this is meant to be a quotation from another article? The quote itself also seems like two sentences have been erroneously joined.
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