Gao and colleagues present a draft Bombax ceiba genome assembly using PacBio sequencing, BioNano optical mapping and Illumina sequencing. From genome assembly, annotation and gene analyses, different methods were applied to scrutinise the quality of the assembly. However, before the acceptance of this manuscript I have some concerns and suggestions:x000Dx000D Major: x000D 1. Authors used a Kmer-based method to estimate the genome size. However, it seems the estimated genome size is smaller than their assembly size. Do authors know what happens here? x000D 2. For contamination checking, do authors believe a visualised GC content plot and the average GC content can tell there is no contamination in the assembly? Why didn't authors use other methods to check, such as BLASTN?x000D 3. From the report, there is a ~150Mb difference between the BioNano consensus maps and the PacBio assembly. Does that mean there are repeat collapses in the PacBio assembly or some genome regions cannot be covered by PacBio reads? How does it look like in the comparison between BioNano consensus maps and the PacBio assembly? Do they align well? x000D 4. This research is more likely a technical and bioinformatics report. Some biological stories, such as important genes, their functions and roles in certain pathways, are missing. How can this research help to improve Bombax ceiba's economic and ecological values?x000D 5. The English in the manuscript needs a further edit_x000D_ Minor: x000D 1. It would be good to specify the version of each tool and detail the parameter settings.x000D 2. It would be good to map the Illumina short reads (DNA) back to the assembly and give a mapping rate to inform the quality of the assembly.x000D 3. L177: It may not be good to say this. Please rewrite. x000D 4. FigureS4 and TableS4 are not informative. May change them to a table to list the stats of the raw BioNano maps.x000D 5. TableS5 may add two more columns: one is for the PacBio assembly, and the other one is for the BioNano optical scaffolding. From the improved table, readers can easily tell the changes before and after BioNano scaffolding.

Level of interest Please indicate how interesting you found the manuscript:
An article whose findings are important to those with closely related research interests

Quality of written English Please indicate the quality of language in the manuscript:
Needs some language corrections before being published

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Authors' response to reviews:

Reviewer: 1 1. For every software, please (if not in the manuscript, then in the supplementary text) provide the non default parameters that were used, if any. Answer: The suggestion has been taken seriously, and the versions and parameters of software used in our study have been provided as one supplementary table. Please see Table S15 in the supplementary file. 2. Line 60 precise the type (SE, PE, MP) and number of cycle of the Illumina sequencing. Answer: We accept the review’s suggestion, and the sequencing strategy (PE) has been added. Please see line 60 in the revised manuscript. 3. Line 106 - I would like some more details about the Illumina based scaffolding. Given that it's a small insert size library (400bp), how helpful was it to scaffold the PacBio assembly. Or do you mean the RNA-Seq data was used to scaffold? If that's the case, what parameters were used and how were multiple mapping case handled? Answer: The sequencing data from the Illumina DNA library (400bp) was aligned against the genome assembly. As the reviewer stated, the small insert size library had relatively limited effects on scaffolding. The analysis was aimed at filling gaps and correcting some sequencing errors of the genome assembly. Sorry for the ambiguity. The sentence has been corrected. Please see line 106 to 108. 4. Line 111 - I would replace the title there to: Evaluation of the completeness of the genome assembly gene space. BUSCO only look at the gene space. For the completeness of the genome assembly, one would need different metrics, such as FRC (feature response curve) or any quantification of the reads that do not align to the final assembly. Answer: We take the review’s suggestion, and the title has been changed to “Evaluation of the completeness of the genome assembly gene space”. 5. Line 226 - There's a typesetting error in "financially" Answer: We accepted the review’s suggestion, and this error has been corrected in the revised manuscript.

Discretionary comments 1. Line 114 - Have you analysed the reads that do not align to the genome, what are these? Answer: Unfortunately, we did not analyze the RNA reads which could not align to the genome assembly, and we did not know the details on these reads. 2. Line 116 - Have you checked what the 5.6% of incomplete BUSCO genes are? Answer: There are 63 missing BUSCO genes and 19 incomplete BUSCO genes in total. We have searched these genes against the OrthoDB database to infer the gene functions. For the 63 missing genes, there are eight genes falling in the category of pentatricopeptide repeat (PPR) superfamily protein, seven genes belonging to protein kinase domain, six genes annotated as transmembrane: Helical, five genes belonging to the tetratricopeptide repeat (TPR)-like superfamily proteins, and five genes annotated with unknown functions. The rest of genes were annotated with a variety of functions, such as peptidyl-prolyl cis-trans isomerase, WD40 repeat, regulator of chromosome condensation (RCC1) family protein, and so on. For the 19 incomplete genes, two genes were characterized as transmembrane: Helical, and two genes were annotated with unknown functions. The other genes were annotated with a variety of functions, such as Zinc finger (C2H2), Reticulon-like protein, and so on. 3. Line 152 - The annotation could be refined using PASA. This would add splice variants to the annotation. Also, the PASA output can be post-processed to identify lncRNA. Many tools now allow for an in-depth analysis of differential transcript expression, differential transcript usage, etc. leveraging from more complete annotations. Answer: As the reviewer suggested, PASA was a useful tool, especially for annotating splice variants. And we will try this software in our future study.

Reviewer: 2 1. Authors used a Kmer-based method to estimate the genome size. However, it seems the estimated genome size is smaller than their assembly size. Do authors know what happens here? Answer: As the reviewer pointed, there was some inconstancy between the size of the genome survey and the genome assembly. As the heterozygosity rate of the B. ceiba genome was 0.88%, heterozygous regions which could not be assembled into consensus sequences might result in larger assembly. Besides, the Kmer-based method was just a preliminary estimation of the genome size. There could be some small deviation from the genome assembly. 2. For contamination checking, do authors believe a visualised GC content plot and the average GC content can tell there is no contamination in the assembly? Why didn't authors use other methods to check, such as BLASTN? Answer: There were two strategies for deducing contaminations by GC content. 1. There were uneven GC contents in the genome. 2. The GC content was constant, but the sequencing depth varied among sequences. BLASTN was also a useful method for assessing contaminations. The suggestion of the reviewer has been taken seriously, and we randomly selected some sequences from the genome assembly and searched the data against the NCBI database of bacteria with BLASTN. No contamination from bacteria was detected. Please check line 88 to 91 in the manuscript. 3. From the report, there is a ~150Mb difference between the BioNano consensus maps and the PacBio assembly. Does that mean there are repeat collapses in the PacBio assembly or some genome regions cannot be covered by PacBio reads? How does it look like in the comparison between BioNano consensus maps and the PacBio assembly? Do they align well? Answer: The BioNano consensus genome map recorded the order of sequence fragments and the gap size between adjacent contigs. The BioNano genome map included gaps (Ns), whereas contigs of the primary PacBio assembly did not contain any gaps. It might be the reason why there was a ~150Mb difference. And the inconstancy between BioNano genome map and genome assembly size was also observed in other species, such as Fragaria vesca [1] and Tribolium castaneum [2]. With the initial PacBio genome assembly as reference, approximately 54 Gb out of 160 Gb BioNano clean data could be assigned to the genome map, and the effective coverage of assembly was about 60 X.

Reference 1. Edger PP, VanBuren R, Colle M, et al. Single-Molecule Sequencing and Optical Mapping Yields an Improved Genome of Woodland Strawberry (Fragaria Vesca) with Chromosome-Scale Contiguity. GigaScience. 2018;7 (2):1-7. doi:10.1093/gigascience/gix124. 2. Shelton JM, Coleman MC, Herndon N, et al. Tools and Pipelines for Bionano Data: Molecule Assembly Pipeline and Fasta Super Scaffolding Tool. BMC Genomics. 2015;16 (1):734.

1. This research is more likely a technical and bioinformatics report. Some biological stories, such as important genes, their functions and roles in certain pathways, are missing. How can this research help to improve Bombax ceiba's economic and ecological values? Answer: As the reviewer suggested, information on important genes and pathways will help to demonstrate the usefulness of our genome data. B. ceiba is an ecologically important plant which could survive in extreme climate conditions. So we calculated the average Ka/Ks values and conducted a branch-site likelihood ratio test to identify positively selected genes which could contribute to the ecological adaptation of B. ceiba. Thirty-six genes were identified, and some annotated genes were reported to be associated with ecological adaption. Please see line 190 to 210 in the revised manuscript.
2. The English in the manuscript needs a further edit Answer: We accepted the reviewer’s suggestion, and the language has been polished by “sees-editing Ltd”. Minor:
3. It would be good to specify the version of each tool and detail the parameter settings. Answer: We accepted the reviewer’s suggestion, and the versions and parameters of software used in our manuscript were provided as one supplementary table. Please see Table S15 in the supplementary file.
4. It would be good to map the Illumina short reads (DNA) back to the assembly and give a mapping rate to inform the quality of the assembly. Answer: We accepted the reviewer’s suggestion, and we have mapped the Illumina short reads (DNA) back to the assembly. A mapping rate of 99.2% was achieved Please check line 106 to 108 in the revised manuscript.
5. L177: It may not be good to say this. Please rewrite. Answer: As the reviewer suggested, we rewrote part of the sentence. As the detail on parameters was very important for this analysis, we kept this information in the revised manuscript. Please check line 178 in the manuscript.
6. FigureS4 and TableS4 are not informative. May change them to a table to list the stats of the raw BioNano maps. Answer: We took the reviewer’s suggestion, and FigureS4 was removed. TableS4 was changed to a table which included statistics of the raw BioNano maps.
7. TableS5 may add two more columns: one is for the PacBio assembly, and the other one is for the BioNano optical scaffolding. From the improved table, readers can easily tell the changes before and after BioNano scaffolding. Answer: We took the reviewer’s suggestion, and this information was added to TableS5. Please check Table S5 in the supplementary file.

Finally, we appreciate much your time in editing our manuscript and the reviewers for their valuable suggestions and comments. We look forward to hear your final decision when it is made.

Thank authors for improving the manuscript and providing a quick response to my comments. Based on the reply I still have some concerns and suggestions.

For my previous question 1: the authors pointed out that heterozygosity may affect the estimation of genome size by using Kmers. Do authors believe <1% heterozygosity rate can lead to ~100 Mb assembly differences (the final assembly is 895Mb)? Is that possible 17-mer underestimated the genome size (I understand that in BGI's paper they used 17-mer to estimate giant panda's genome size. Is 17-mer suitable for B.ceiba? If authors test different kmers, I suppose you will have different results).

For my previous question 2: I appreciate that authors used BLASTN to confirm contaminations. However, shouldn't authors use the non-plant database instead of bacteria? Why did authors randomly select some contigs (how many?) instead of all of them? I understand that using random selection to avoid bias, but since the contamination rate is low (I suppose), you will have less chance to select a contamination contig if you only select a few contigs from the pool.

For my previous question 3: I understand that there are some discrepancies between Bionano consensus maps and the NGS reference. I want to know how many Bionano consensus maps can align to the Pacbio assembly or the total size of the aligned PacBio assembly? Since Bionano gives 1.09Gb consensus maps, does that mean the real genome size of B.ceiba is around 1.09 Gb? Does that mean the Pacbio assembly is still underestimated? For the 'Ns' added through Bionano scaffolding, it can be easily checked.

New questions: 1. why did authors change the final assembled genome size from 869Mb to 895Mb, but didn't change any stats, is anything wrong with the previous calculation? 2. From the density of Bionano label, it seems it is low. Which enzyme did authors use to generate the Bionano data? Is that Nt. BspQI?

Level of interest Please indicate how interesting you found the manuscript:
An article whose findings are important to those with closely related research interests

Quality of written English Please indicate the quality of language in the manuscript:
Acceptable

Declaration of competing interests Please complete a declaration of competing interests, considering the following questions: Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Do you hold any stocks or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Do you hold or are you currently applying for any patents relating to the content of the manuscript? Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript? Do you have any other financial competing interests? Do you have any non-financial competing interests in relation to this paper? If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests.

I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.
I agree to the open peer review policy of the journal.

Authors' response to reviews: Reviewer #2: 1. For my previous question 1: the authors pointed out that heterozygosity may affect the estimation of genome size by using Kmers. Do authors believe <1% heterozygosity rate can lead to ~100 Mb assembly differences (the final assembly is 895Mb)? Is that possible 17-mer underestimated the genome size (I understand that in BGI's paper they used 17-mer to estimate giant panda's genome size. Is 17-mer suitable for B.ceiba? If authors test different kmers, I suppose you will have different results). Answer: As the reviewer suggested, we reanalyzed the genome size with other K-mers (19-mer and 21-mer). The estimated genome size was 835 Mb (19-mer) and 821 Mb (21-mer), respectively. The results did not dramatically depart from the genome size estimated with 17-mer (809 Mb). And our previously study of flow cytometry also suggested that the genome size of B. ceiba was approximately 800Mb (2C =1.55±0.03pg) [1]. The heterozygosity rate of B. ceiba genome was 0.88%. As pointed by many researchers, genomes with heterozygosity rate higher than 0.5% are considered as highly heterozygous [2]. Assembling highly heterozygous diploid genomes is a substantial challenge, and heterozygous regions could not be assembled into consensus may result in larger assembly [2]. So we concluded that the highly heterozygous genome of B. ceiba might be the main reason why there were ~100 Mb differences between the estimated genome size and the final assembly.

1. For my previous question 2: I appreciate that authors used BLASTN to confirm contaminations. However, shouldn't authors use the non-plant database instead of bacteria? Why did authors randomly select some contigs (how many?) instead of all of them? I understand that using random selection to avoid bias, but since the contamination rate is low (I suppose), you will have less chance to select a contamination contig if you only select a few contigs from the pool. Answer: We accepted the reviewer’s suggestion, and we searched all sequences of the genome assembly against the NCBI nucleotide collection (Nt) with BLASTN with E-value < 1e-5 and sequence identity > 70%. In total, 2494 significant hits were achieved. The top-hit species were Theobroma cacao and Gossypium species, comprising more than 69% of the hits (1733 hits). Only five hits from four non-plant species (Psyllidae sp., Trioza eugeniae, Diptacus sp., and Dichorragia nesimachus) were detected, which suggested there was no potential contamination from non-plant species in the genome of Bombax ceiba. Please see line 89-95 in the revised manuscript and Table S4 in the supplementary file.

2. For my previous question 3: I understand that there are some discrepancies between Bionano consensus maps and the NGS reference. I want to know how many Bionano consensus maps can align to the Pacbio assembly or the total size of the aligned PacBio assembly? Since Bionano gives 1.09Gb consensus maps, does that mean the real genome size of B.ceiba is around 1.09 Gb? Does that mean the Pacbio assembly is still underestimated? For the 'Ns' added through Bionano scaffolding, it can be easily checked. Answer: Approximately 64.3 Gb (2023 individual maps) out of 160 Gb BioNano clean data could be mapped to the Pacbio assembly, and the average molecule coverage depth of the genome map was 27 × (Please check line 106-108 in the revised manuscript). The final genome assembly scaffolded by BioNano mapping contained 386 gaps (25,395,219 bp in total). The larger genome consensus map (1.09 Gb) should mainly be contributed to redundancies because of the high genome heterozygosity.

New questions: 1. why did authors change the final assembled genome size from 869Mb to 895Mb, but didn't change any stats, is anything wrong with the previous calculation? Answer: We are sorry for this matter. During the initial submission, we made a mistake and took the contig size (869Mb) as the assembled genome size due to negligence. So we changed the size of the final genome assembly to 895Mb (the scaffold size) in the revised manuscript. We appreciate very much for this comment. 2. From the density of Bionano label, it seems it is low. Which enzyme did authors use to generate the Bionano data? Is that Nt. BspQI?

Answer: We used Nt. BspQI enzyme in the BioNano mapping procedure. This information has been added to the revised manuscript. Please see line 98-99.

Finally, we appreciate the quick, detailed, useful comments and suggestions from the reviewers for improving our manuscript.

Reference

1. Zhou ZL, Xiong Z, Huan-Cheng MA, et al. Genome Sizes of Two Bombacaceae Plants. Journal of West China Forestry Science. 2014;43 (2):97-101. (in Chinese)
2. Kajitani R, Toshimoto K, Noguchi H, et al. Efficient De Novo Assembly of Highly Heterozygous Genomes from Whole-Genome Shotgun Short Reads. Genome Research. 2014;24 (8):1384-95.

Thank authors for giving detailed responses to my concerns. I think this manuscript is ready to be accepted.

Level of interest Please indicate how interesting you found the manuscript:
An article whose findings are important to those with closely related research interests

Quality of written English Please indicate the quality of language in the manuscript:
Acceptable

Declaration of competing interests Please complete a declaration of competing interests, considering the following questions: Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Do you hold any stocks or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Do you hold or are you currently applying for any patents relating to the content of the manuscript? Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript? Do you have any other financial competing interests? Do you have any non-financial competing interests in relation to this paper? If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests.

I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.
I agree to the open peer review policy of the journal.