Copy Number Variation: Methods and Clinical Applications

Round 1

Reviewer 1 Report

Authors provide information on various methods of CNV detection, bioinformatic tools for processing NGS data, and potential biomedical applications of CNV identification. But, most importantly, they present information on tools for annotation and classification of identified CNVs, as well as the list of the most popular databases for assessing the pathogenetic significance of chromosomal aberrations.

Nowadays it is evident that CNVs are not only a source of pathogenic but also a normal variation. In some cases a chromosomal mutation in a patient with neurodevelopment disorder may be mistakenly classified as pathogenic, actually being a polymorphic variant that is quite common in a population. An incorrect assessment of the pathogenetic significance may later become a reason for pregnancy termination if the fetus carries a similar CNV as a sick child. In several cases, detected chromosomal mutations have not yet been described in the literature, or identified patients with the mutation are too few to assess its pathogenic effect. Most of the detected CNVs remain in the VOUS category. Therefore, the issue of CNV interpretation is still relevant. It is necessary to evaluate the detected rearrangements through the use of at least several databases. The information presented in the article will be useful for decision by clinicians, genetic counsellors, and laboratory geneticists.

I have no comments on the text of the publication, however, some links do not work correctly, in particular, a link for ECARUCA source (another website opens). Also, I can't access the Chromosomal Variation in Man database.

Author Response

Dear reviewer,

We are sending a revised version of the manuscript with ID applsci-1070011, entitled “Copy number variation: methods and clinical applications”. We have carefully read all the reviewer's suggestions and found them accurate and valuable. Our responses to the reviewer’s comments are provided in the attached document (Response to reviewer 1.docx) and changes were incorporated into the updated version of the manuscript.

We hope that the updated version of the manuscript is now suitable for publication in the Applied Sciences journal.

Best wishes,

Ondrej Pös

Author Response File: Author Response.docx

Reviewer 2 Report

I consider that the review is interesting and is significant contribution specially to the ones that are starting in the field and to summarize the main methods for CNV detection, however, there are a couple of new methods to detect CNV from 2GS and 3GS, such as Canvas SPW and MFCNV, that are not mentioned.  Further, the English should be revised, there are several paragraphs that are quite confusing and impairs a fluid read.

Some specific suggestions:

Page 1, Line 34-36: Please re-formulate this sentence, it is quite confusing: “Currently, the lower limit for a variant length to be classified as CNV is considered to be 50 bps, but this value has been gradually decreasing from 1000 kbs, due to continuous methodological progress.”

Page 1, line 42: Authors should not cite an unpublished manuscript “(for more detailed reasoning see Pös et al. 2020; Manuscript currently submitted to Biomedical Journal).

Legend of Figure 1: I consider that the title is not well suited for the content. Authors state that are methods for the assessment of CNV, but some are not specific for CNV detection but for sequencing in general and the methods for CNV detections are not fully described. Consider changing to a more suitable title.  For instance: “Hallmarks in CNV history. The 20th century was characterized by the development of methods that allowed genome-wide and high-resolution CNV detection at the beginning of the 21st century”.

Also, in figure 1, the references should agree with the rest of the text. Otherwise, is difficult for the reader to localize the reference.

Page 2, line 53: “If the CNV affects a coding region, disruption of gene function or alteration of gene dosage may occur”. This sentence must be reformulated, I understand that the authors say it in the next sentence, but it gives the idea that only the coding regions can cause diseases which is not true. Check these papers: https://0-doi-org.brum.beds.ac.uk/10.1093/hmg/ddv259 and https://0-doi-org.brum.beds.ac.uk/10.1016/j.critrevonc.2020.103113

Page 2, line 69-72: “However, the age of high throughput technologies is are coupled with an increasing amount of generated data.(…)”

For a clear reading I suggest to re-write, for instance: “Despite the value of high throughput technologies for CNV detection, these technologies generate an increasing amount data, imposing the need to continuously improve bioinformatics software and  guidelines to help clinicians to handle these high amounts of data”

Page 2, lines 77-79: The authors need to revise the English of the sentence.

Page 3, lines 90 – 93: Quite confuse sentence. Reformulate. Try to create shorter sentences or eliminate unnecessary information to make the read more fluid and less confusing. 

Page 3, lines 133-134: “A tremendous improvement in the screening of unbalanced structural variation, including CNVs …”. I suggest that authors briefly explain what is structural variation and what is the relation with CNV.

Page 4, lines 181-182:” There are three main strategies for 2GS-based CNV analysis: including whole-genome, whole-exome, and targeted sequencing.” I suggest also a brief explanation of how CNV can be detected with 2GS as authors did with PCR and hybridization-based methods previously.

Page 5, lines 194- 195: “… 3GS offered by Oxford Nanopore Technologies provides pocket-sized, low-cost devices…”. Authors should reformulate. As it is, seems that only Oxford Nanopore Technologies provide 3GS which is not true. Further, there are certainly more updated references for this technology.

Table 1 and Table 2: I consider that would be valuable for readers if these tools are free or not and which computational requirements they have (for instance, if are available for windows or if only work in linux)

Page 6, line 254-255: Regarding the sentence “CNVs have been studied in neuropsychiatric, developmental, and cardiovascular diseases [57].” I suggest including specific and updated references for each condition referred.

Author Response

Dear reviewer,

We are sending a revised version of the manuscript with ID applsci-1070011, entitled “Copy number variation: methods and clinical applications”. We have carefully read all the reviewer's suggestions and found them accurate and valuable. Our responses to the reviewer’s comments are provided in the attached document (Response to reviewer 2.docx) and changes were incorporated into the updated version of the manuscript.

We hope that the updated version of the manuscript is now suitable for publication in the Applied Sciences journal.

Best wishes,

Ondrej Pös

Author Response File: Author Response.docx