Construction of a new chromosome-scale, long-read reference genome assembly for the Syrian hamster, Mesocricetus auratus

  1. R. Alan Harris
  2. Muthuswamy Raveendran
  3. Dustin T. Lyfoung
  4. Fritz J Sedlazeck
  5. Medhat Mahmoud
  6. Trent M. Prall
  7. Julie A. Karl
  8. Harshavardhan Doddapaneni
  9. Qingchang Meng
  10. Yi Han
  11. Donna Muzny
  12. Roger W. Wiseman
  13. David H. O’Connor
  14. Jeffrey Rogers

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Background

The Syrian hamster ( Mesocricetus auratus ) has been suggested as a useful mammalian model for a variety of diseases and infections, including infection with respiratory viruses such as SARS-CoV-2. The MesAur1.0 genome assembly was generated in 2013 using whole-genome shotgun sequencing with short-read sequence data. Current more advanced sequencing technologies and assembly methods now permit the generation of near-complete genome assemblies with higher quality and greater continuity.

Findings

Here, we report an improved assembly of the M. auratus genome (BCM_Maur_2.0) using Oxford Nanopore Technologies long-read sequencing to produce a chromosome-scale assembly. The total length of the new assembly is 2.46 Gbp, similar to the 2.50 Gbp length of a previous assembly of this genome, MesAur1.0. BCM_Maur_2.0 exhibits significantly improved continuity with a scaffold N50 that is 6.7 times greater than MesAur1.0. Furthermore, 21,616 protein coding genes and 10,459 noncoding genes are annotated in BCM_Maur_2.0 compared to 20,495 protein coding genes and 4,168 noncoding genes in MesAur1.0. This new assembly also improves the unresolved regions as measured by nucleotide ambiguities, where approximately 17.11% of bases in MesAur1.0 were unresolved compared to BCM_Maur_2.0 in which the number of unresolved bases is reduced to 3.00%.

Conclusions

Access to a more complete reference genome with improved accuracy and continuity will facilitate more detailed, comprehensive, and meaningful research results for a wide variety of future studies using Syrian hamsters as models.

Article activity feed

  1. GigaScience

    report

    Reviewer name: Yang Zhou (revision 1)

    The authors have resolved most of my comments. However, I am still confused about the gap in the Pilon step from the information in Table 1. In the table, I could read that the assembly length of "Flye + Pilon" is 2,383,228,608 bp, and the ungapped legnth is 2,383,226,373 bp, so the gap length is 2,383,228,608 - 2,383,226,373 = 2,235 bp. Because in the "Flye" version the assembly length is equal to the ungapped legnth, this means that gaps are introduced after Pilon correction. Level of Interest Please indicate how interesting you found the manuscript: Choose an item. Quality of Written English Please indicate the quality of language in the manuscript: Choose an item. 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.

  2. GigaScience

    Findings

    ****Reviewer name: Yang Zhou****

    The authors have resolved most of my comments. However, I am still confused about the gap in the Pilon step from the information in Table 1. In the table, I could read that the assembly length of "Flye + Pilon" is 2,383,228,608 bp, and the ungapped legnth is 2,383,226,373 bp, so the gap length is 2,383,228,608 - 2,383,226,373 = 2,235 bp. Because in the "Flye" version the assembly length is equal to the ungapped legnth, this means that gaps are introduced after Pilon correction. Level of Interest Please indicate how interesting you found the manuscript: Choose an item. Quality of Written English Please indicate the quality of language in the manuscript: 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.

  3. GigaScience

    Syrian

    Reviewer name: Derek Bickhart (revision 2)

    The authors have addressed all of my remaining concerns. Level of Interest Please indicate how interesting you found the manuscript: Choose an item. Quality of Written English Please indicate the quality of language in the manuscript: Choose an item. 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.

  4. GigaScience

    Background

    Reviewer name: Derek Bickhart (revision 1)

    Summary: In this revision, the authors have addressed most of my major concerns with the manuscript. More details must be provided in two sections of the manuscript based on new details provided by the authors. However, these concerns could feasibly be addressed in revision. Line 124: While the authors have provided an explanation for the sequencing of different target fragment length library preparations, I do not see any results that suggest that one particular preparation was more efficient than the others. This is particularly important given the prevalence of four experimental runs of varying dataset sizes that were uploaded to the cited Biosample accession on SRA. Currently, the metadata provided for that Biosample and its associated experiments is lacking, and one cannot easily distinguish which experiment resulted from different target length preparations. A discursive analysis is not required here, but a statement that provides limited data supporting the authors' preference for library prep is necessary. Line 301: I believe that the authors misinterpreted the comment on this section in my last review. I requested the proportion of sequence identity differences between assemblies due to INDELs, not assembly gaps. Residual INDELs are still a major problem in polished assemblies that may impact gene annotation. Figure 1 caption: Given the new k-mer genome size estimation analysis provided by the authors, it does not make sense to use the total length of the MesAur1.0 assembly here. I believe that the authors should choose a genome size estimate that seems most reasonable (from the two options provided) and then use that as the basis for NG50 comparisons. Otherwise, are they conceding that the MesAur1.0 assembly size is the full length of the Syrian Hamster sequence-accessible genome? Level of Interest Please indicate how interesting you found the manuscript: Choose an item. Quality of Written English Please indicate the quality of language in the manuscript: Choose an item. 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.

  5. GigaScience

    Abstract

    This work has been peer reviewed in GigaScience (see paper), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer name: Derek Bickhart

    Summary: In this manuscript, Harris et al. detail the methods they used to create a new reference genome for the Syrian hamster, which is an important model for respiratory disease pathogens. They used several different sequencing technologies to generate the contigs and scaffolds for their new assembly, and achieved a relatively continuous end product. The analysis is suitable for the "genome report" style format (with one omission detailed below in my comments); however, the manuscript suffers from some awkward phrasing and grammar errors in the results and methods. I list my comments below in the relative order in which I encountered them in the manuscript. Since the authors did not provide line numbers in their submission, I provide my comments as a block listing of questions/suggestions/critiques. Section titled "oxford nanopore long-read sequencing": The description of the shearing is awkward. I recommend revising the first sentence to state that the genomic DNA isolates were sheared to three lengths (without providing these lengths in the sentence). In subsequent sentences, provide the lengths in situ with the methods used to prepare them. Also, it is unclear why three different fragment lengths were used here for oxford nanopore sequencing. Given that these fragment lengths are relatively similar in size (e.g. not disparate lengths similar to recent ultra-long nanopore read preps of >100kb), it would be very helpful to the reader if justification was given for this approach. Section titled "Genome assembly": This entire paragraph is awkwardly phrased with numerous past- or present-tense changes. Additionally, the reference to the Pilon polisher needs to be cited, and details need to be provided on what settings were used for Pilon polishing (it is often recommended to correct only indels and to omit gap-filling) and how many iterations of polishing were used. Details are missing on how BioNano optical maps were generated, and what DNA was used as input in the process. Also, what software was used to compare BioNano optical maps, and with what settings? Finally, it appears that the RNA-seq data used by NCBI for annotation was used in another study. Citation to that study would be required so that the reader is aware that the data resulted from different individuals other than the reference individual sequenced in this analysis. Section titled "Assembly Comparisons": What is the expected c-value of the Syrian Hamster genome? Also, what is the karyotype count? Are any of the chromosomes metacentric or acrocentric? Were any satellite regions identified and annotated in this assembly? Finally, I would have preferred that assembly comparisons be conducted with feature response curves, such as those produced by the program "FRC_align" as this provides a useful metric to assess assembly "correctness" by length. Section titled "Transcript and protein alignments and annotation comparisons": How many INDELs were identified in the alignments of RNA-seq transcripts to the BCM_Maur_2.0 assembly? Was this count different from those discovered in the short read assembly? Section titled "Interferon type 1 alpha gene cluster": Were there any gaps that spanned the gene cluster or flanked it? Level of Interest Please indicate how interesting you found the manuscript: Choose an item. Quality of Written English Please indicate the quality of language in the manuscript: Choose an item. 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.

  6. Version published to 10.1101/2021.07.05.451071v4 on bioRxiv
  7. Version published to 10.1101/2021.07.05.451071v3 on bioRxiv
  8. Version published to 10.1101/2021.07.05.451071v2 on bioRxiv
  9. ScreenIT

    SciScore for 10.1101/2021.07.05.451071: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Data from this individual are available in NCBI BioProject PRJNA705675
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    The two smaller length fragment libraries were sheared using Covaris gTube and the 30kb targeted size library was fragmented with Diagnode Megarupter 3, all following manufacturer’s recommendations.
    Covaris
    suggested: None
    Base-calling was done onboard the PromethION instrument with the use of neuronal network based software (Oxford Nanopore Technologies, UK)
    PromethION
    suggested: (PromethION, RRID:SCR_017987)
    The Flye assembler v2.8.1 [18] was used to generate the initial de novo genome assembly.
    Flye
    suggested: (Flye, RRID:SCR_017016)
    Pilon uses high quality Illumina reads mapped to an assembly to correct errors arising from the lower quality ONT sequencing data.
    Pilon
    suggested: (Pilon , RRID:SCR_014731)
    Quality assessment: To assess the quality of our assembly compared to the previous MesAur1.0 we used Quast v5.0.2 [20] together with MUMmer v3.23 [21] and Assemblytics [22].
    Quast
    suggested: (QUAST, RRID:SCR_001228)
    MUMmer
    suggested: (MUMmer, RRID:SCR_018171)
    In addition, the Illumina reads from the original reference (NCBI SRA SRR413408) were mapped to our assembly and the MesAur1.0 reference using BWA v0.7.17 [23] and Quast was used to obtain discordant pair statistics.
    BWA
    suggested: (BWA, RRID:SCR_010910)
    The software Benchmarking Universal Single-Copy Orthologs (BUSCO) v3.0.2 [24] was used for quality assessment of the genome assembly.
    Benchmarking Universal Single-Copy Orthologs
    suggested: (BUSCO, RRID:SCR_015008)
    BUSCO was performed using the OrthoDB v9 (odb9
    BUSCO
    suggested: (BUSCO, RRID:SCR_015008)
    OrthoDB
    suggested: (OrthoDB, RRID:SCR_011980)
    The MesAur1.0 genome assembly is available in the NCBI database under BioProject PRJNA77669 (GenBank accession GCA_000349665.1).
    NCBI
    suggested: (NCBI, RRID:SCR_006472)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  10. Version published to 10.1101/2021.07.05.451071v1 on bioRxiv