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Thank you for addressing comments/suggestions from the previous academic editor. The manuscript is now accepted for publication in PeerJ.
The authors addressed all my concerns and I am happy with the revised manuscript.
Already answered previously. All good.
Already answered previously. All good.
The authors have addressed my comments.
The authors have addressed my comments.
The authors have addressed my comments.
Please address issues raised by the reviewers, in particular a discussion of haplotypes and their phasing in relation to the results, and the clarifications in the method description and exposition as requested.
With respect to additional references, I refer to PeerJ policy. I do think that adding a paragraph or two to the beginning of the introduction, placing the work in a bigger context within genome biology (and not focussed on tools), could potentially very nicely round out the manuscript and make it more accessible for readers who are not method developers.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
Tolstoganov et al report SpLitteR, a tool to resolve assembly graphs using linked reads. Overall the work is interesting, novel, and well presented. The main shortcoming that I would like to see addressed is the absence of specific considerations regarding haplotypes and their phasing, even though it's actually mentioned as one of the goals/results in the abstract. Yet this is a critical aspect in diploid genome assembly. It is not clear how haplotypes are impacted (for instance in Fig1), apparently they were not directly evaluated or results are not reported/discussed. It is also unclear how the algorithm could be integrated in tools that perform haplotype-resolution (presumably at a later stage).
Minor:
"Afterward, long fragments within the containers are sheared into shorter fragments and sequenced." > the barcoding step should also be mentioned
"However, even though linked-reads result in more contiguous assemblies than assemblies based on non-linked short reads, all these tools generate rather fragmented assemblies of large genomes and metagenomes." > references needed, e.g. https://www.nature.com/articles/s41586-021-03451-0
Isn't CTGA miscolored in figure 1 (shouldn't it be ACT or ACTG)
It isn't clear if homopolymer compression is a feature of the tool or needs to be performed separately (if so, it is not specified how)
line 132: bold
In line with the scope of the journal. Rigorous work.
Impact and novelty are significant. Support data well presented, conclusions well-stated
Comments:
- The abstract and introduction are well-crafted, providing a clear overview.
- The manuscript acknowledges limitations effectively, notably the challenges with HiFi-based assemblies in repeat-rich areas, where repeats can extend beyond the length of HiFi reads, impacting the continuity of diploid genome assemblies due to shared long identical regions between two haplomes. Tell-seq data, though leading to more contiguous or fragmented assemblies when used alone, offers a solution. Please cite review article: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-021-02328-9
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
- The process of aligning barcoded reads, achieving uniqueness with a k-mer length of k=31 for edge sequences, and its relation to the underlying graph theory is well explained, though more integration with graph theoretical principles could enhance understanding. Please refer article for readers: https://www.nature.com/articles/s41467-023-36689-5.
**PeerJ Staff Note:** It is PeerJ policy that additional references suggested during the peer-review process should only be included if the authors agree that they are relevant and useful.
- How well does this method work in highly polymorphic regions?
- The results section is well-supported with detailed information on read length, number of reads, coverage, and post-assembly data quality presented in informative tables. The use and evaluation of tools for graph assembly are thoroughly described.
- The method section's introduction (lines 83-87) disrupts the flow, with suggestions to align the explanation of branching more closely with Figure 1 or to integrate it into lines 89-99.
- References to the “manufacturer’s user guide” are missing in the data preparation section (lines 114, 121).
- Lack of comparison between results from multiplex de Bruijn graphs (mDBG) and other types of assembly graphs derived from GFAs.
- Unclear descriptions are noted in several areas: conversion from a standard GFA to mDBG encoding (lines 136-138), and the notation of graph edges and vertices (lines 140-145), recommending adherence to standard notations as in "Introduction to Algorithms" (CLRS). The description of rel_thr and abs_thr parameters (lines 182-183) is also vague, with only values provided.
The experimental design seems appropriate, well-defined, relevant, and meaningful.
The findings are valid and novel.
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