An assessment of Nano-RECall: Interpretation of Oxford Nanopore sequence data for HIV-1 drug resistance testing
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Abstract
Introduction
Oxford Nanopore Technologies (ONT) offer sequencing with low-capital-layout sequencing options, which could assist in expanding HIV drug resistance testing to resource limited settings. However, sequence analysis remains time time-consuming and reliant on skilled personnel. Moreover, current ONT bioinformatic pipelines provide a single consensus sequence that is not equivalent to Sanger sequencing, as drug resistance is often detected in mixed populations. We have therefore investigated an integrated bioinformatic pipeline, Nano-RECall, for seamless drug resistance of low read coverage ONT sequence data from affordable Flongle or MinION flow cells.
Methods
We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n=46) and b) homebrew integrase (IN) (n=21). We investigated optimal read-depth by assessing the coefficient of variation (CV) of nucleotide proportions for various read-depths; and between replicates of 400 reads. The agreement between Sanger sequences and ONT sequences were assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations.
Results
The coefficient of variation of ONT minority variants plateaued after a read depth of 400-fold implying limited benefit of additional depth and replicates of 400 reads showed a CV of ∼6 % for a representative position. The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1-99.4%) for PR-RT and 99.6% (95% CI: 99.4-99.7%) for INT. Drug resistance mutations did not differ for 21 IN sequences; 16 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT sequences, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both.
Conclusions
The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource limited settings.
Article activity feed
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10480310.
This review is the result of a Live Review organized and hosted by PREreview and JMIR Publications on September 16, 2022. The call was joined by 10 people, including reviewers, preprint authors, and facilitators.
Summary
The authors of this study present a novel integrated bioinformatic pipeline called Nano-RECall custom fit for Oxford Nanopore Technologies (ONT) sequencing—which offers sequencing options that are more affordable with respect to standard Sanger Sequencing/RECall, that can be used to efficiently and more affordably identify drug-resistant cases of HIV. This pipeline for ONT is presented as an alternative to other existing pipelines which do not meet all the specifications …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10480310.
This review is the result of a Live Review organized and hosted by PREreview and JMIR Publications on September 16, 2022. The call was joined by 10 people, including reviewers, preprint authors, and facilitators.
Summary
The authors of this study present a novel integrated bioinformatic pipeline called Nano-RECall custom fit for Oxford Nanopore Technologies (ONT) sequencing—which offers sequencing options that are more affordable with respect to standard Sanger Sequencing/RECall, that can be used to efficiently and more affordably identify drug-resistant cases of HIV. This pipeline for ONT is presented as an alternative to other existing pipelines which do not meet all the specifications compared to Sager's RECall. Some of the clear advantages of the Nano-RECall pipeline include the fact that the software is openly available, it can run on different operating systems (i.e., Windows, Linux, and Mac), and it has Nanopore specific error correction. To test their new method, the authors used the same HIV 1 subtype C PCR amplicons to compare the outputs of Sanger sequencing using the standard RECall software to the outputs of ONT sequencing using their Nano-RECall bioinformatic pipeline. The comparison revealed that 97.8% of the mutations were detected by both methods, with only a few detected by one but not the other method (true in both directions). Even with some limitations which were discussed by the authors (e.g., the small number of samples tested with only one subtype), this approach is a promising solution, particularly as it is presented as an easier & more cost-effective solution. Below are some comments and feedback collected during the call which we hope will be useful to the authors to improve their manuscript and the delivery of their findings.
Evidence and Examples: Concerns and Constructive feedback
For someone who is not familiar with the standard RECall pipeline it would be beneficial to provide more information about it in the methods section so to help the reader make an informed comparison and interpretation of the results.
It may be helpful, if they are available, to include data from users who have used the pipeline, and perhaps include an additional comparison with the currently available pipeline used for ONT sequencing.
It would be helpful to provide information about the file type and format needed to be fed into the pipeline would be helpful to a reader and potential user of the methodology.
In the introduction the Sanger RECall pipeline is described as one that is complicated as it requires many steps, it requires experts to be used, and is expensive. It would be helpful to present a clear comparison throughout the paper, but especially in the discussion between RECall and Nano-RECall across these characteristics.
Below are other general suggestions and recommendations to improve on the clarity of the work presented.
The reviewers suggest checking that the way figures are introduced in the narrative of the results matches and is aligned with the order in which the figures are presented to make it easier for the reader to refer to the figures while reading the results.
Remove "a)" from Figure 1 as the figure has only one panel.
We suggest increasing the size of labels and numbers in Figures 1 and 2 for easier readability.
Please adjust colors used in Figure 3 to accommodate readability from colorblind community (some advice offered in these posts https://www.ascb.org/science-news/how-to-make-scientific-figures-accessible-to-readers-with-color-blindness, https://www.nature.com/articles/d41586-021-02696-z).
Other points and final remarks
The reviewers appreciated the fact that the authors took time to discuss the comparison of their new methodology with current state of the art approaches, as well as the fact that the software is available on GitHub and open for feedback from the community.
Several authors of the preprint were present in the call and contributed to the discussion. The organizing team is grateful to all the participants of the PREreview + JMIR Publications's Live Review and in particular to the subject matter expert, Dr. George Kolostoumpis and Dr. Susan Egbert for their contribution to the discussion, and to the preprint authors who engaged in this type of collaborative review.
Competing interests
The author of this review is a member of the PREreview team and facilitated the Live Review. They synthesized the notes from the Live Review discussion into this review and also contributed with a few suggestions for the preprint authors.
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