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Os\u00f3rio, ICVS\/3B's - PT Government Associate Laboratory, University of Minho, Braga, 22 Mar 2021, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-42\/v1#referee-response-42794","order":0,"name":"referee-response-42794","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Richard Pearson<\/b>; \n<i>Posted: 06 Jul 2021<\/i>; We thank the reviewers for the extremely positive and supportive feedback. In their comments and suggestions both reviewers have well captured the spirit of this data resource and of the large collaborative network behind it. We are pleased to submit detailed responses and a revised version of the manuscript that addresses their comments.  \n<i><b>1.1) Increasing the accessibility of the table listing polymorphisms in supplementary data. The authors do provide the data in VCF and zarr files, which are not very user friendly nor allow a fast search of a specific polymorphism. We understand that developing a web interface for this purpose would be a challenge beyond this research article but possibly exporting the VCF file data into tables that could be available in online repositories.<\/b><\/i>  We thank the reviewer for this important feedback on how to increase the reach of this resource. Since the publication of this article, we have been working on an initial web interface that allows users to navigate some aspects of the data: please see https:\/\/www.malariagen.net\/apps\/pf6. The current version mainly focuses on epidemiologically relevant data and emphasises the community behind the project and at the moment doesn\u2019t provide access to the genomic variation information, which will require further work.  Of course accessibility is a relative criteria and as such it requires balancing out different priorities. In the past we have provided tabular versions of the data (www.malariagen.net\/data) but the benefits have been very limited. For example, handling multiallelic and non-SNP variations requires somewhat arbitrary encoding decisions that significantly affect the simplicity and intuitiveness of the tabular format. Increasing the sample size has made these variations more common (e.g. in this release there are about 50% non-SNP variants and 50% multiallelic variants) to the point that there was no real advantage in maintaining the format. The decision of primarily utilising the VCF format comes from the recognition that these files are the standard de facto in the genomic community, which in turn has developed a large ecosystem of tools to handle them: please see the README at ftp:\/\/ngs.sanger.ac.uk\/production\/malaria\/pfcommunityproject\/Pf6\/Pf_6_README_20191010.txt for some examples, e.g. to subset the data.  However we agree this might still be limiting for some use cases and we are working towards a more integrated solution. As an example of our direction of travel, please see&nbsp;https:\/\/malariagen.github.io\/vector-data\/landing-page.html, which presents some simplified data access workflows for the MalariaGEN \n<i>Anopheles gambiae<\/i> 1000 Genomes Project.  \n<b><i>1.2) Add to the supplementary file 4, describing the drug resistance markers genotype, the PfMDR1 N86Y. This SNP is a well-known modulator of antimalarial response and considered a risk factor for the treatment of artemether-lumefantrine.<\/i><\/b>  We recognise that there is growing evidence of the role of \n<i>PfMDR1<\/i>&nbsp;N86Y in artemether-lumefantrine resistance. In particular, multiple studies have shown that lumefantrine appears to select for N86. Despite that, WHO still reports markers of resistance to lumefantrine as \u201cYet to be validated\u201d (p. 22 - \n<b>https:\/\/www.who.int\/publications\/i\/item\/9789240012813<\/b>). In this release, supplementary file 4 only contains validated markers so it would be inconsistent to add the markers. However, we will consider adding putative markers in future releases where appropriate.  \n<i><b>1.3) Add the ID of the genes most mentioned in the main article. The gene ID (PF3D7_xxxxxxx), is provided in supplementary file 7, but to clarify the reader, we recommend to add it also in the main article when first describing the genes.<\/b><\/i>  We have implemented the recommendation and added gene IDs every time a gene is mentioned for the first time in the manuscript version 2.  \n<i><b>1.4) In the results section, when describing gene amplification and different sets of breakpoints, the authors describe complex rearrangements that have not been observed before in Plasmodium species. In regards to pfmdr1 duplication events has been described to vary in size while spanning different genes in different parasites1,2,3,4. In a genome walking like approach, it has been described different amplicon sizes containing the pfmdr1 in clinical isolates from Southeast Asia where they also investigated if the type (i.e., which genes are included) and size of the amplicon influence drug susceptibility phenotypes5.<\/b><\/i>  The complex rearrangements that have not been observed before which we were referring to here are \u201cdup-trpinv-dup\u201d rearrangements that to the best of our knowledge have only previously been described in human data (see ref 58). This complex and large structural rearrangement involves a triplicated segment embedded within a duplication, in which the triplicated segment is inverted. We recognise that the original wording in the text was ambiguous and we\u2019ve replaced \u201ccomplex rearrangements\u201d with an explicit description of the event.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-42\/v2#referee-comment-4687","order":1,"name":"referee-comment-4687","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.17752.r42796, Didier Menard, Malaria Genetics and Resistance Unit, Parasites and Insect Vectors Department, Institut Pasteur, Paris, 25 Mar 2021, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-42\/v1#referee-response-42796","order":2,"name":"referee-response-42796","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Richard Pearson<\/b>; \n<i>Posted: 06 Jul 2021<\/i>; We thank the reviewers for the extremely positive and supportive feedback. In their comments and suggestions both reviewers have well captured the spirit of this data resource and of the large collaborative network behind it. We are pleased to submit detailed responses and a revised version of the manuscript that addresses their comments.  \n<i><b>2.1) Sample collection - P. falciparum samples investigated are not from systematic sampling collections dedicated to this study but rather from multiple studies conducted by groups with different objectives and from heterogeneous populations (patients living in malaria endemic areas, travelers, etc.). I think this issue should be discussed in the manuscript.<\/b><\/i>  Thanks for raising this point. On one hand, the heterogeneity of sampling approaches offers a unique opportunity to investigate questions in a variety of epidemiological settings in a systematic way. Specifics of each study are provided in ftp:\/\/ngs.sanger.ac.uk\/production\/malaria\/pfcommunityproject\/Pf6\/Pf_6_partner_studies.pdf and users of the resource can contact individual investigators for further details. At the same time, we agree that this can also act as a confounder in some analysis, which is why we\u2019ve devoted significant time to the curation of the dataset to make it \u201canalysis ready\u201d.  As suggested, we have amended the manuscript in version 2 to include the considerations above in the paragraph: \u201cSamples were collected by independent groups that were operative at a given time and in a given place with distinct objectives; while care needs to be taken when interpreting results spanning multiple years and geographical settings (e.g. aggregated trends of drug resistance prevalence), this heterogeneity also allows for the exploration of a wide range of epidemiological and transmission settings.\u201d  \n<b><i>2.2) Likewise, the long time period covering the samples collection (2002\u20132015) is also a major bias which can alter the final results.<\/i><\/b>  This is an important point in particular for interpreting drug resistance results, and one we explicitly bring out in the paragraph: \u201cNote that samples were collected over a relatively long time period (2002\u201315) during which there were major changes in global patterns of drug resistance, and that the sampling locations represented in a given year depended on which partner studies were operative at the time. To alleviate this problem, we have also divided the data into samples collected before and after 2011 (Supplementary Data; Supplementary table 10), but temporal trends in aggregated data should be interpreted with due caution.\u201d. Following the reviewer\u2019s suggestion, we have now stressed this point further in our reply to point (2.1) above.  \n<b><i>2.3) I guess that all samples were collected from symptomatic patients seen at health facilities level? Unfortunately, this makes that data presented capture only P. falciparum populations infected this population. With the rise of new technologies, I am wondering whether the MalariaGEN consortium could investigate samples collected from asymptomatic individuals and explore the genomic profiles of this hidden reservoir but representing the major parasite biomass?<\/i><\/b>  Asymptomatic infections are indeed an incredibly significant reservoir that needs to be explicitly considered to achieve a complete and accurate picture of the transmission landscape. The development of new technologies has begun to dig deeper and deeper in this area and initial results seem to be very encouraging that good quality data can indeed be obtained from asymptomatic and\/or low parasitemia subjects. MalariaGEN would certainly be supportive of this kind of effort and we have indeed active collaborations with partners exploring these questions. To the best of our knowledge, though, some of these methodologies are still of limited sensitivity and in part experimental and will require further work in order to be deployed on the large scale required by this scientific question, but that is certainly an area for future investigation.  \n<b><i>2.4) I am aware that the authors have performed a difficult and complex exercise by providing high quality genomic data and comprehensive description of their data for a large audience. The major challenge that is not addressed in the manuscript is how these important data can be translated into concrete actions in the field by health providers.<\/i><\/b>  This data resource represents a clear step towards the ultimate objective of translating genomic surveillance outputs into actionable actions, although it is fair to say that this is a long journey with many different components. The ability for multiple groups to share data, to analyse it using standardised methods, and to make it readily accessible is the foundation for translational impact to reach maturity.  In the discussion we highlighted a series of future translational directions which have been and will be facilitated by resources like this one (and future ones) but it is certainly true that these results require careful interpretation due to the caveats highlighted in the paper and by the reviewer, which inevitably limit their impact. At the same time this dataset does create a systematic framework to enact and contextualize future discoveries of that nature and, indirectly, contributes to them.  Ultimately, the practical value for malaria control will be greatly enhanced by the progressive acquisition of longitudinal time-series data and their integration with other sources of epidemiological data which will allow control programmes to monitor the impact of their interventions on the parasite population in near real time.  \n<b><i>2.5) Last comment regarding the database. It will be helpful to provide for each sample\/genome sequence, the location (country) and the date of collection.<\/i><\/b>  This information is included in the \u201cSample provenance and sequencing metadata\u201d file available at the resource page https:\/\/www.malariagen.net\/resource\/26","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-42\/v2#referee-comment-4688","order":3,"name":"referee-comment-4688","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"The sequencing, analysis, informatics and management of the Community Project are supported by Wellcome through Sanger Institute core funding (098051), a Strategic Award (090770\/Z\/09\/Z) and the Wellcome Centre for Human Genetics core funding (203141\/Z\/16\/Z), by the MRC Centre for Genomics and Global Health which is jointly funded by the Medical Research Council and the Department for International Development (DFID) (G0600718; M006212), and by the Bill & Melinda Gates Foundation (OPP1204628). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}