"Don't look up" is so real and not just in the media

We talked about BBC, i.e., Black-bone chicken and how they have dispersed across Asia after sequencing the genomes of Kadaknath chicken. The focus of the earlier post was mostly on the history and what can be inferred regarding the possible history based on genetics. Yet, one of the technically challenging tasks performed by Shinde et al., 2023 was the resolution of the structural organisation of the Fm locus. When the study was published on 22nd June 2023, almost all the previous literature strongly supported the *Fm_2 as the correct arrangement of the Fm locus. Hence, it seemed trivial to establish this based on long-read sequencing. However, we were in for a big surprise.

The Surprise

The surprise came on 06th December 2023 with the publication of a "chromosome-level genome assembly for the Silkie chicken" as part of a Communications Biology paper. Communications Biology proclaims itself as an "open access journal". It is published by the Nature Portfolio to cover "high-quality research, reviews and commentary in all areas of the biological sciences". The journal has an impact factor in the range of 5 to 6 and could be comparable to some of the journals in the Frontiers portfolio, such as the following:

1. Frontiers in Bioengineering and Biotechnology
2. Frontiers in Cell and Developmental Biology
3. Frontiers in Cellular and Infection Microbiology
4. Frontiers in Cellular Neuroscience
5. Frontiers in Chemistry
6. Frontiers in Endocrinology
7. Frontiers in Immunology
8. Frontiers in Microbiology
9. Frontiers in Molecular Biosciences
10. Frontiers in Nutrition
11. Frontiers in Pharmacology
12. Frontiers in Plant Science
13. Frontiers in Public Health
14. Public Health Reviews

Now, what distinguishes Communications Biology from these journals? Established in 2018 as a sister journal to Communications Physics and Communications Chemistry, the journal is aimed at "providing a new open access option for biologists while applying less stringent criteria for impact and significance than the Nature-branded journals, including Nature Communications." This journal aims to fill the gap between the different Nature portfolio journals. 

Returning to the surprise of 6th December, the new genome assembly of the Silkie chicken presented by CAU (China Agricultural University) claims that the *FM_1 is the correct arrangement of the Fm locus. The Chinese CAU was formed in 1995 and should not be confused with the Central Agricultural University at Lamphelpat, Imphal (formed by an act of parliament in 1992). CAU is among the top 350 global universities in the world university rankings. In the agriculture and forestry ranking, CAU is one of the top universities ranked at least in the top 10. With 5 shared first authors and 3 shared corresponding authors, the study represents a large body of work of great interest to the chicken genomics community. As soon as it became clear that the results of the CAU_Silkie genome were contradictory to those of Shinde et al., 2023, the obvious questions were whether Shinde et al. got the result wrong? If they got it wrong, what step did they go wrong in and why?

The fear of being wrong

The CAU_Silkie genome manuscript was submitted to CommBio on 3rd August 2022 and Accepted only on 21 November 2023. This again suggests a very thorough review spanning more than 1 year. To be more precise, it is 475 days or 1 year, 3 months, 18 days or 15 months, 18 days. The author list consists of scientists from the UK, Australia, Canada, and multiple institutes in China. Again, this suggests a big multi-national study by serious big genomics groups. At first sight, it appeared that Shinde et al. had published a wrong result. Publishing a wrong result is always a big nightmare for any researcher, as it suggests many things and could be interpreted in many more ways.

On a side note, the first version of the Shinde et al. pre-print appeared on bioRxiv on August 01, 2022. The revised version of this pre-print was posted on March 06, 2023, and accepted at Frontiers in Genetics on June 05 2023. The review time of ~3 months at Frontiers is 1/5th the time spent by the CAU_Silkie genome manuscript in review. Could the Shinde manuscript receive a detailed enough review in 3 months? Although the names of the reviewers are not listed, the journal notes, "Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work." This suggests more than 1 person may have reviewed the CAU_Silkie genome article. While the Frontiers article lists the names of the three reviewers, the number of person-hours spent reviewing the article seems much higher for the CAU_Silkie genome manuscript.

We quickly analysed the references cited by the CAU_Silkie genome paper using the RIS file downloaded from the Communications Biology website. Of the 133 references listed, 124 have the publication year in the PY field of RIS format. 

•       1 PY  - 1979
•       1 PY  - 1994
•       1 PY  - 1995
•       1 PY  - 1996
•       1 PY  - 1998
•       5 PY  - 1999
•       1 PY  - 2000
•       2 PY  - 2001
•       1 PY  - 2003
•       6 PY  - 2004
•       3 PY  - 2005
•       1 PY  - 2007
•       4 PY  - 2008
•       4 PY  - 2009
•       4 PY  - 2010
•       4 PY  - 2011
•       4 PY  - 2012
•       3 PY  - 2013
•       9 PY  - 2014
•       6 PY  - 2015
•       7 PY  - 2016
•       9 PY  - 2017
•       8 PY  - 2018
•      14 PY  - 2019
•       9 PY  - 2020
•       8 PY  - 2021
•       5 PY  - 2022
•       1 PY  - 2023

The paper from 2023 is the PNAS paper on the "Evolutionary analysis of a complete chicken genome". Quantifying the number of papers cited based on the journal in which it is published shows most cited articles are from Bioinformatics and Nucleic Acids Res.

•       1 JO  - Agric. Gene
•       1 JO  - Am. J. Physiol.
•       1 JO  - Avian Pathol.
•       1 JO  - Biochem. Mol. Biol. Int.
•       1 JO  - BMC Biol.
•       1 JO  - BMC Biotechnol.
•       1 JO  - BMC Genet.
•       1 JO  - Cell
•       1 JO  - Cell Metab.
•       1 JO  - Cell Res.
•       1 JO  - Cell Syst.
•       1 JO  - Chromosoma
•       1 JO  - Cytogenet Genome Res.
•       1 JO  - Dev. Comp. Immunol.
•       1 JO  - Endocrine
•       1 JO  - Endocrinology
•       1 JO  - Endocr. Rev.
•       1 JO  - Front. Agr. Sci. Eng.
•       1 JO  - Front. Biosci.
•       1 JO  - Front. Physiol.
•       1 JO  - G3 (Bethesda)
•       1 JO  - Gene
•       1 JO  - Immunobiology
•       1 JO  - Immunol. Today
•       1 JO  - Int. J. Endocrinol.
•       1 JO  - J. Biol. Chem.
•       1 JO  - J. Cell Biochem.
•       1 JO  - J. Hered.
•       1 JO  - Mol. Cell Endocrinol.
•       1 JO  - NAR Genom. Bioinforma.
•       1 JO  - Nat. Genet.
•       1 JO  - Nat. Metab.
•       1 JO  - Nat. Protoc.
•       1 JO  - Nat. Rev. Rheumatol.
•       1 JO  - Paleobiology
•       1 JO  - R. Soc. Open Sci.
•       1 JO  - Sci. China Life Sci.
•       1 JO  - Trends Endocrinol. Metab.
•       2 JO  - BMC Bioinforma.
•       2 JO  - Dev. Dyn.
•       2 JO  - Front. Immunol.
•       2 JO  - Methods Mol. Biol.
•       2 JO  - Mol. Immunol.
•       2 JO  - PLoS Comput. Biol.
•       3 JO  - Gen. Comp. Endocrinol.
•       3 JO  - Immunogenetics
•       3 JO  - Nat. Biotechnol.
•       3 JO  - Nat. Commun.
•       3 JO  - Nat. Methods
•       3 JO  - PLoS ONE
•       3 JO  - Poult. Sci.
•       3 JO  - Proc. Natl. Acad. Sci. USA
•       3 JO  - Science
•       4 JO  - BMC Genomics
•       4 JO  - Mol. Biol. Evol.
•       4 JO  - PLoS Genet.
•       5 JO  - J. Immunol.
•       6 JO  - Genome Biol.
•       6 JO  - Nature
•       8 JO  - Nucleic Acids Res.
•       9 JO  - Bioinformatics

Quantifying the number of papers by the author's name reveals most papers cited are from Kaufman, J. 

•       1 AU  - Afanassieff, M.
•       1 AU  - Aharoni, T.
•       1 AU  - Akash, M. S. H.
•       1 AU  - Akiba, Y.
•       1 AU  - Ashwell, C.
•       1 AU  - Ashwell, C. M.
•       1 AU  - Baek, M.
•       1 AU  - Bagatto, B.
•       1 AU  - Bailey, T. L.
•       1 AU  - Balakrishnan, C. N.
•       1 AU  - Baldwin-Brown, J. G.
•       1 AU  - Baudouin-Gonzalez, L.
•       1 AU  - Beck, S.
•       1 AU  - Bed’hom, B.
•       1 AU  - Bennett, C.
•       1 AU  - Bennett, C. E.
•       1 AU  - Black-Pyrkosz, A.
•       1 AU  - Bolger, A. M.
•       1 AU  - Bornelov, S.
•       1 AU  - Borodovsky, M.
•       1 AU  - Borst, S. E.
•       1 AU  - Boswell, T.
•       1 AU  - Botero-Castro, F.
•       1 AU  - Brocht, D. M.
•       1 AU  - Bruna, T.
•       1 AU  - Bruneau, G.
•       1 AU  - Butter, C.
•       1 AU  - Camacho, C.
•       1 AU  - Carroll, S. B.
•       1 AU  - Carvajal, A. K.
•       1 AU  - Chakraborty, M.
•       1 AU  - Chang, S.
•       1 AU  - Chan, P. P.
•       1 AU  - Chazara, O.
•       1 AU  - Cheng, H.
•       1 AU  - Chen, L.
•       1 AU  - Chiang, C.
•       1 AU  - Cline, D. L.
•       1 AU  - Concepcion, G. T.
•       1 AU  - Courcelle, E.
•       1 AU  - Covey, S. D.
•       1 AU  - Czerwinski, S. M.
•       1 AU  - Dakovic, N.
•       1 AU  - Dalman, M. R.
•       1 AU  - Dharmayanthi, A. B.
•       1 AU  - Dong, K.
•       1 AU  - Durand, N. C.
•       1 AU  - Durbin, R.
•       1 AU  - Eddy, S. R.
•       1 AU  - Einat, P.
•       1 AU  - Emerson, J. J.
•       1 AU  - Erben, H. K.
•       1 AU  - Erickson, C. A.
•       1 AU  - Eshdat, Y.
•       1 AU  - Fakiola, M.
•       1 AU  - Fan, J.
•       1 AU  - Faraco, C. D.
•       1 AU  - Faraut, T.
•       1 AU  - Farkašová, H.
•       1 AU  - Feng, C.
•       1 AU  - Feng, X.
•       1 AU  - Figuet, E.
•       1 AU  - Firtina, C.
•       1 AU  - Flynn, J. M.
•       1 AU  - Friedman, J. M.
•       1 AU  - Galtier, N.
•       1 AU  - Ghurye, J.
•       1 AU  - Glavas, M. M.
•       1 AU  - Goel, M.
•       1 AU  - Goto, R. M.
•       1 AU  - Gouet, P.
•       1 AU  - Grabherr, M. G.
•       1 AU  - Grant, C. E.
•       1 AU  - Greiff, K.
•       1 AU  - Hansen, N. F.
•       1 AU  - Hateren, A.
•       1 AU  - Heller, D.
•       1 AU  - Henry, J.
•       1 AU  - Hincke, M. T.
•       1 AU  - Hjellnes, V.
•       1 AU  - Hoefs, J.
•       1 AU  - Hoff, K. J.
•       1 AU  - Holt, J.
•       1 AU  - Horev, G.
•       1 AU  - Hron, T.
•       1 AU  - Huang, Z.
•       1 AU  - Hu, J.
•       1 AU  - Imsland, F.
•       1 AU  - Ivashkiv, L. B.
•       1 AU  - Jain, C.
•       1 AU  - Jeffares, D. C.
•       1 AU  - Jiao, W. B.
•       1 AU  - Johnson, J.
•       1 AU  - Johnson, M.
•       1 AU  - Jones, C. D.
•       1 AU  - Kaiser, P.
•       1 AU  - Kalliolias, G. D.
•       1 AU  - Kalvari, I.
•       1 AU  - Kaspers, B.
•       1 AU  - Kieffer, T. J.
•       1 AU  - Kim, D.
•       1 AU  - Kim, J.
•       1 AU  - King, M. D.
•       1 AU  - Kolbe, D. L.
•       1 AU  - Kolmogorov, M.
•       1 AU  - Koren, S.
•       1 AU  - Lamont, S. J.
•       1 AU  - Lee, G. H.
•       1 AU  - Liaqat, A.
•       1 AU  - Lin, Y.
•       1 AU  - Liu, P.
•       1 AU  - Liu, Q.
•       1 AU  - Liu, S.
•       1 AU  - Loehlin, D. W.
•       1 AU  - Lohse, M.
•       1 AU  - Lomsadze, A.
•       1 AU  - Londraville, R. L.
•       1 AU  - Long, A. D.
•       1 AU  - Lovell, P. V.
•       1 AU  - Love, M. I.
•       1 AU  - Lowe, T. M.
•       1 AU  - Manni, M.
•       1 AU  - Mann, K.
•       1 AU  - Marcais, G.
•       1 AU  - McMillan, L.
•       1 AU  - McMurtry, J. P.
•       1 AU  - Mellouk, N.
•       1 AU  - Mesa, C. M.
•       1 AU  - Milne, S.
•       1 AU  - Monget, P.
•       1 AU  - Moreno, A.
•       1 AU  - Morgenstern, B.
•       1 AU  - Morin, V.
•       1 AU  - Nabholz, B.
•       1 AU  - Nawrocki, E. P.
•       1 AU  - Noble, W. S.
•       1 AU  - Nolan, L. K.
•       1 AU  - O’Connor, R. E.
•       1 AU  - Okimoto, R.
•       1 AU  - Pačes, J.
•       1 AU  - Paggi, J. M.
•       1 AU  - Pajer, P.
•       1 AU  - Park, C.
•       1 AU  - Parks-Dely, J. A.
•       1 AU  - Pastor, M. V.
•       1 AU  - Pereira, S.
•       1 AU  - Pevzner, P. A.
•       1 AU  - Phillippy, A. M.
•       1 AU  - Pierce, B. G.
•       1 AU  - Pitel, F.
•       1 AU  - Pontarotti, P.
•       1 AU  - Prokop, J. W.
•       1 AU  - Pyl, P. T.
•       1 AU  - Qu, F.
•       1 AU  - Rehman, K.
•       1 AU  - Reyes-Grajeda, J. P.
•       1 AU  - Rhie, A.
•       1 AU  - Robert, X.
•       1 AU  - Rohde, F.
•       1 AU  - Romero, A.
•       1 AU  - Rustad, T.
•       1 AU  - Salzberg, S. L.
•       1 AU  - Sato, K.
•       1 AU  - Schneeberger, K.
•       1 AU  - Seppey, M.
•       1 AU  - Sharp, P. J.
•       1 AU  - Shaw, I.
•       1 AU  - Sherman, B. T.
•       1 AU  - Sherman, R. M.
•       1 AU  - Shiina, T.
•       1 AU  - Siedler, F.
•       1 AU  - Slizyte, R.
•       1 AU  - Smith, J.
•       1 AU  - Solinhac, R.
•       1 AU  - Staines, K.
•       1 AU  - Sun, Z.
•       1 AU  - Takahashi, K.
•       1 AU  - Takimoto, T.
•       1 AU  - Taouis, M.
•       1 AU  - Tartaglia, L. A.
•       1 AU  - Taylor, R. L.
•       1 AU  - Thulien, K. J.
•       1 AU  - Tian, M.
•       1 AU  - Tian, X.
•       1 AU  - Tilak, M. K.
•       1 AU  - Tixier-Boichard, M.
•       1 AU  - Tregaskes, C. A.
•       1 AU  - Usadel, B.
•       1 AU  - Vaz, S. A.
•       1 AU  - Vingron, M.
•       1 AU  - Waddington, D.
•       1 AU  - Walker, B. A.
•       1 AU  - Wang, J. R.
•       1 AU  - Wang, K.
•       1 AU  - Wang, M. S.
•       1 AU  - Wang, P.
•       1 AU  - Wang, Y.
•       1 AU  - Warren, W. C.
•       1 AU  - Wedepohl, K. H.
•       1 AU  - Wong, G. K.
•       1 AU  - Xie, Q.
•       1 AU  - Yin, Z. T.
•       1 AU  - You, Z.
•       1 AU  - Yuan, J.
•       1 AU  - Yuen, Z. W.
•       1 AU  - Zdobnov, E. M.
•       1 AU  - Zhang, G.
•       1 AU  - Zhang, Y.
•       1 AU  - Zhao, S.
•       1 AU  - Zhu, F.
•       1 AU  - Zoorob, R.
•       2 AU  - Anders, S.
•       2 AU  - Dunn, I. C.
•       2 AU  - Elleder, D.
•       2 AU  - Haas, B. J.
•       2 AU  - Huber, W.
•       2 AU  - Moon, D. A.
•       2 AU  - Stanke, M.
•       2 AU  - Sun, H.
•       2 AU  - Veniamin, S. M.
•       2 AU  - Zhang, H.
•       2 AU  - Zhang, J.
•       3 AU  - Dorshorst, B.
•       3 AU  - Friedman-Einat, M.
•       3 AU  - Li, H.
•       3 AU  - Magor, K. E.
•       3 AU  - Miller, M. M.
•       3 AU  - Seroussi, E.
•       4 AU  - Kaufman, J.

Of these references, 1 to 30 are first cited in the Introduction (30). 31-83 in the Results (53), 84-91 in the Discussion (8), 92 to 132 in the Methods (41) and 133 is cited in the Code availability (1) section. The results section has the highest number of first citations. 

In what way are the results different?

The CAU_Silkie genome abstract describes the result: "We also provide whole-genome methylation and genetic variation maps, and resolve a complex genetic region that may contribute to fibromelanosis in these animals."

Figure 1b of the CAU_Silkie genome paper describes the Fm locus result: "The genomic collinearity for inverted duplication associated with fibromelanosis and the Hi-C heatmap of Chr20. Arrows of different colors represent the clip direction. The density curves at each end represent sequencing coverage." The order in the figure is DUP1-> <-DUP2 <-INT <-DUP1 - DUP2->. 

Compare this with the 3 scenarios possible:

*FM_1: DUP1-> <-DUP2 <-INT <-DUP1 - DUP2->

*FM_2: DUP1-> <-DUP2 - DUP1-> INT-> DUP2->

*FM_3: DUP1-> INT-> DUP2-> <-DUP1 - DUP2->

The Shinde et al. study claims to establish *FM_2 as the correct arrangement and is in concordance with previous studies published in Plos Genetics: Dorshorst (2015) and PLoS ONE: Dharmayanthi (2017). While the Journal of Heredity: Dorshorst (2010) paper maps the location of Fm, nothing is known about the structure of this region then. The GigaScience: Sohn (2018) paper does try to solve the structure of the Fm locus and argues that scenario 1 is potentially correct. However, this GigaScience paper is completely ignored in the CAU_Silkie study as the breed used is Yeonsan Ogye, not Silkie.

In addition to the main figure, the Supplementary Figure S11 of the CAU_Silkie genome manuscript has a cartoon depiction of "The possible rearrangement hypothesis of hyperpigmentation". 

Interestingly, here, *FM_1 is defined as DUP1-> <-DUP2 - DUP1-> INT-> DUP2->. This corresponds to the FM2 of the Dorshorst (2015) paper.

In the same figure, *FM_2 is defined as DUP1-> <-DUP2 - <-INT <-DUP1 - DUP2->. This corresponds to the *FM_1 of the Dorshorst (2015) paper.

This redefinition seems unnecessarily confusing, and the motivation for such renaming is hard to comprehend. 

Some more details about the dataset and the genome assembly:

1. The CAU_Silkie_1.0 genome available on NCBI (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_033088195.1/) has a total size of 1080553668 compared to the 1,080,256,408 mentioned in Supplementary Table 1. What is the source of the 297260 bp difference between these two values?
2. Although the CAU manuscript states, "DNA from the same female Silkie bird was used to generate PacBio, Nanopore sequencing libraries", the genome assembly does not have a W chromosome assembled. Shouldn't the complete genomic sequence include the W chromosome?
3. Data from two projects are linked to the CAU_Silkie genome paper. The project PRJNA805080 contains the genomics data, and PRJNA827662 contains the transcriptomic data. The genomics data consists of paired-end Illumina re-sequencing data from 15 samples, data from one female individual consisting of 2 libraries of PromethION, 3 libraries of HI-C (Illumina NovaSeq 6000) and 3 libraries of Sequel II. All the fastq read headers lack library details except for SRR17968808. Why are the headers stripped of the original information? Does SRA need a way to validate the data? This is a severe flaw in the procedure of sequencing data deposition in these archives.

PacBio HiFi sequencing of Gal gallus: adult female silkie - Blood

>gnl|SRA|SRR17981950.1.11 Biological (Biological) m64066_200930_085914 483 Data size(G) 

>gnl|SRA|SRR17981951.1.11 Biological (Biological) m64061_201030_024601 494 Data size(G)

>gnl|SRA|SRR17981952.1.11 Biological (Biological) m64082_201009_073749 410 Data size(G)

Hi-C sequencing of Gal gallus: adult female silkie - Breast muscle

>gnl|SRA|SRR17968807.1.11 Biological (Biological) 200826_A00682_0423_BH5YHJDSXY 4.1 Data size(G)
>gnl|SRA|SRR17968808.1.1A00262:510:HCWVKDSXY:2:1101:1018:1031 Biological (Biological) 200911_A00869_0294_AHCW57DSXY 102 Data size(G)
>gnl|SRA|SRR17968809.1.11 Biological (Biological) 200918_A00262_0510_BHCWVKDSXY 5.1 Data size(G)

Nanopore sequencing of Gal gallus: adult female silkie - Blood

>gnl|SRA|SRR17968711.1.11 Biological (Biological) PAG02028_e86ed44e 44 Data size(G)
>gnl|SRA|SRR17968712.1.11 Biological (Biological) PAG02064_2db1aec4 31 Data size(G)

While these minor issues with the dataset and the way it is shared are common in genomics papers, the contradictory result concerning the structural organisation of the Fm locus is a major difference.

The relief of not being wrong

One of the first things we did after the 6th December surprise was re-analysing all the data from the Shinde et al., 2023 manuscript. To our great relief, our conclusions seemed to be correct and well-founded. However, our understanding may be wrong, or the CAU_Silkie genome manuscript interpretation may need to be corrected. 

The Fm locus region assembly is a challenging task. For instance, the earlier genomes of Silkie2 (GCA_024679325.10) and Silkie3 (GCA_024653025.1) reporting the De Novo Assembly of 20 Chicken Genomes in the journal Molecular Biology and Evolution (Impact Factor: 10.7, 4 out of 52 in Evolutionary Biology), 2022 did not manage to assembly the Fm locus and resolve its correct structure. The genome assembly of Yeonsan Ogye (GCA_002798355.1) generated by Sohn et al. (2018) and published in Gigascience (Impact Factor: 9.238) does not provide a complete assembly of this region and cannot identify which of the 3 possible scenarios is correct.

No wonder the CAU silkie paper published in Communications Biology (Impact Factor: 6.548) has the following bold proclamation at the end of the paper: "In our case, the high-quality Silkie chicken assembly solves FM traits which had not been investigated by large-scale chicken pan-genome assemblies, especially based on the second-generation whole-genome sequencing data, and insufficient third-generation data."

As I write this text, 39 Chicken (Gallus gallus) genomes are available on the NCBI genome page. Several were published last year (2023) and used long-read sequencing technologies. More chicken genomes are expected to be published soon as the costs decrease, and the technology becomes more widely available.

1. Four of the assemblies (GCA_016699485.1, GCA_016700215.2, GCA_027408225.1, and GCA_027557775.1) are from the VGP (Vertebrate Genome Project). 
2. Four high-quality assemblies of indigenous (Hu, Piao, Wuding and Daweishan) chicken genomes were published in a single study by Wu et al., 2024.
3. Twenty assemblies are from a single study by Li et al., 2022 and span all major breeds of chicken: Asil, Naked Neck, Fayoumi, White Plymouth Rock, Daweishan, Liyang,  Chahua, Houdan, Thailand Gamefowl, Langshan, White Leghorn (White_Leghorn_2 and White_Leghorn_3), Rhode Island Red (Rhode_Island_Red_2 and Rhode_Island_Red_3), Cornish (Cornish_2 and Cornish_3), Tibetan chicken (Tibetan_chicken_2_zangji and Tibetan_chicken_3_TB), Two Silkie (Silkies_3_SK and Silkies_2_wuji). Most of the samples used in this study are provided by China Agricultural University, proving that CAU is the undisputed leader in chicken genomics. Two samples are from Chittagong Veterinary and Animal Sciences University (CVASU), Bangladesh. 
4. Two Illumina short-read-only assemblies (one male and one female) generated using MaSuRCA v. 4.0.8 from Chattogram Veterinary and Animal Sciences University (CVASU) are not linked to any published articles.

Genome Assembly	DUP1	DUP2	INT	FLANK1	FLANK2	Breed
GCA_030914265.1	124368	178823	421602	735439	502763	Piao
GCA_024686315.1	126610	170999	410657	507381	509386	Langshan
GCA_024686285.1	126677	170938	412850	510730	502380	Thailand Gamefowl
GCA_027408255.1	126695	170999	412990	408641	502266	Ross
GCA_016700215.2	126701	170978	412877	504844	502221	Cross of Broiler mother + white leghorn layer father
GCA_016699485.1	126714	171020	413030	504992	502627	Cross of Broiler mother + white leghorn layer father
GCA_025370635.1	126725	170844	416113	503748	501855	Tibetan chicken
GCA_024653045.1	127158	170923	411693	508443	501395	Houdan
GCA_024687005.1	127320	177882	419518	542738	557884	Cornish
GCA_000002315.5	127379	170815	412533	503216	502498	Red Jungle Fowl
GCA_034509845.1	127411	171032	412747	504320	502225	Hailanhe
GCA_024686295.1	127422	181556	414953	543593	539657	Chahua
GCA_027557775.1	127446	170939	412896	504096	501984	Ross
GCA_027408205.1	127446	170936	412847	493490	502012	Cobb
GCA_024652985.1	127464	173787	412750	503891	502322	Rhode Island Red
GCA_024686355.1	127464	170955	411332	503580	501959	Asil
GCA_030979905.1	127471	170964	412882	503757	501648	Hu
GCA_024686465.1	127485	170954	412751	504173	502916	Naked Neck
GCA_030849555.1	127499	170977	412841	503785	502889	Daweishan
GCA_030914275.1	127500	170949	412831	504572	502165	Wuding
GCA_024652995.1	127503	170862	412765	503950	502148	White Leghorn
GCA_024653035.1	127510	170921	412874	507907	500932	Cornish
GCA_024206055.2	127516	171029	412362	505101	502739	Huxu
GCA_024686275.1	127516	170844	412531	504882	502030	Fayoumi
GCA_027408225.1	127538	170982	412918	499931	502231	Cobb
GCA_034769225.1	127602	170971	412375	503714	502886	GG1M (isolate)
GCA_034769275.1	127651	171017	412409	504978	503879	GG1F (isolate)
GCA_024679905.1	127802	180936	419275	532420	516919	White Leghorn
GCA_024653025.1	129169	1763	412215	504060	502166	Silkie
GCA_024679415.1	129678	170969	411404	496876	511457	White Plymouth Rock
GCA_024679395.1	131654	186473	430346	544670	564581	Tibetan chicken
GCA_024679355.1	132866	175664	438903	534081	516172	Daweishan
GCA_024679375.1	133534	180358	431227	516130	521784	Liyang
GCA_024679765.1	139881	182361	410841	540793	503976	Rhode Island Red
GCA_024679325.1	154503	189852	411884	528345	557780	Silkie
GCA_002798355.1	171519	240450	413946	507857	507596	Yeonsan Ogye
GCA_033088195.1	255007	342004	412392	504829	502201	Silkie
GCA_034509865.1	265295	341998	412333	713469	502903	Silkie
GCA_034509885.1	288497	343770	412770	503902	502371	Lueyang

Matters Arising: A potential mechanism for self-correction

It is one thing to know that "you think you are right" and entirely another when "you are actually right". Science is supposed to be self-correcting and has robust mechanisms in place to ensure such self-correction. One of the existing processes for the self-correction of science is the framework for peers to perform post-publication reviews through comments and criticism of peer-reviewed publications. Enough has been written about the limitations of the peer-review process already. However, post-publication review is one of the solutions to overcome the shortcomings of peer review. Such post-publication peer review (PP PR) can be anonymous, as it happens on websites like PubPeer and the like. Another form of PP PR is by publishing comments or "Letters to the Editor" in the same journal that published the original article or in a related journal. While such comments and letters have existed in one form or another, a more formalised peer-reviewed version of the process has been missing or at least not very effective.

One initiative by Springer that makes some positive strides in this direction is the "Matters Arising" article type. It is described as "Formal post-publication commentary on published papers can involve either challenges or clarifications of the published work and may, after peer review, be published online as Matters Arising, usually alongside a Reply from the original Nature authors." The system in place for these types of articles has matured over the years as the editors and the scientific community have become more familiar with this setup. For instance, the current version of the offering states that "Matters Arising are exceptionally interesting and timely scientific comments and clarifications on original research papers published within the past 18 months in Nature.". This hard deadline of 18 months seems to have been worked out after looking at the trend in the past few years. 

Contributions from India are becoming more common

While Matters Arising type articles are a type of "Research Article", they are rarely published by Indian authors. It may have something to do with a mindset of avoiding challenging authority, "Mai-Baap Sarkar". Speaking truth to power is not alien to Indians. After all, Mahatma Gandhi popularised satyagraha (literally, "truth-force"). Yet, we all know how that turned out not just for Gandhi but also for Martin Luther King Jr. The Matters Arising that we could identify from India are listed below:

• BMC Infectious Diseases (From Department of Community Health, Christian Medical College, Vellore, India)
• Nature Communications (From Department of Biostatistics, St John’s Medical College, Bengaluru, India)
• Communications Biology (Department of Biological Sciences, IISER Bhopal, Bhauri, Madhya Pradesh, India)

Do Matters Arising articles provide a resolution or just present both sides of the debate?

The last one we list is the result of Sharma et al., trying to claim that *Fm_2 is the correct scenario. In the journal Communications Biology, ~7000 articles have been published compared to 23 Matters Arising (including the replies). This works out to less than half a percent of articles (0.33%). So even globally, Matters Arising type articles are rare. After all, nobody wants to be a Galileo. In Scientific Reports, 62 Matters Arising, compared to 231641 articles, is a very small fraction. We compiled the list of these submissions and their replies to see how we stand compared to other Matters Arising articles. The time between the publication of the original article and the submission of the Matters Arising article (Received Days) and the time between the publication of the original article and the acceptance of the Matters Arising article were plotted. See below.

1. Matters Arising Received - Original article date = Received Days
2. Matters Arising Accepted - Original article date = Accepted Days

Two clusters can be seen. One consists of Matters Arising that were submitted within ~100 days of the original articles, and another cluster of articles submitted more than ~300 days after the original article. While every Matters Arising has a Reply published soon after, one does not. At 448 days, the Sharma et al. manuscript does have the longest review time for articles submitted within 100 days of the original article. The longer review time does give some confidence that the science behind it has been evaluated in detail. Compared to the short review time of Frontiers, the multiple rounds of review by multiple experts do ensure greater confidence. 

Possible challenges or sources of error in Shinde et al.:

In this additional scrutiny, what factors were considered in greater detail?

1) Raw read data errors: Could the HDPs, haplotype-consistent tiling paths simply result from sequencing errors.

2) Use other datasets (from other papers) and quantify junction read counts to evaluate if heterozygote/homozygote individuals are used.

3) Genome assemblies (39 available): Do the results remain the same with different genome assemblies? The following assemblies were used: GRCg6a, bGalGal1.mat.broiler.GRCg7b, CAU_Silkie_1.0.

4) Read mapper: bwa mem vs NGMLR vs minimap2. Could the HDPs be an artefact of the read mapper used?

5) Error in counting read support: javarkit vs IGV vs samtools vs custom Python scripts??

For some more aspects, we carefully parse the Reply to Sharma et al.

1) The second line of the reply is, "Our work1 has assembled a Silkie genome (CAU_Silkie) using a composite approach, resolving in one fell swoop the complex genomic variation of the fibromelanosis (Fm) trait and identifying a large number of important genes related to metabolism, immunity, and reproduction in birds."

The phrase "one fell swoop" appears in Macbeth (Act 4, Scene 3) when Macduff learns that Macbeth has murdered his wife and children. In shock and grief, he cries out:

"All my pretty ones?
Did you say all? O hell-kite! All?
What, all my pretty chickens and their dam
At one fell swoop?"

Here, "fell" means fierce, deadly, or cruel, and "swoop" refers to the way a bird of prey, like a falcon or kite, suddenly attacks. Macduff compares Macbeth to a "hell-kite" (a ruthless predator) and laments how his family was wiped out in a single, merciless attack—"one fell swoop."

Over time, the phrase evolved to mean doing something all at once, often with dramatic or decisive effect. Notably, Macduff says, "all my pretty chickens"

2) The first line of the second paragraph is, "The structural variation involved in Fm includes not only genomic duplications but also inversions, especially since the individual assembled in our work is a heterozygote (Fm/fm).". In contrast to the original article, in this reply, it is claimed that the individual assembled is a heterozygote. This is particularly important as Sharma et al. claim the individual is a homozygote. 

Having made this claim, the authors state, "The validity of our results is supported by several solid lines of evidence."

(a) First evidence is the results of read mapping to the wild-type (*N) and mutant type (*Fm_1) structures. 

(b) The second evidence is "the String Graph and contigs from haplotype assembly". 

Based on this reasoning, the authors conclude, "All the evidence suggests that our results are correct."

Once the evidence is presented, the reply turns to the problematic evidence from Sharma et al.,. "Nagarjun et al.4 mentions some ‘key pieces of evidence’ that argue there is something wrong with our assembly, but they are all quite problematic.". 

In the sentence:

mentions some ‘key pieces of evidence’ that argue there is something wrong with our assembly,

the single quotes around ‘key pieces of evidence’ are used for one of the following reasons:

1. Emphasis or distancing: The writer might be signaling that they are quoting someone else's exact words or that they are not fully endorsing the phrase. It can imply skepticism, irony, or a different interpretation than the literal meaning.

2. Scare quotes: These are quotation marks used around a word or phrase to indicate that it’s being used in a non-standard, unusual, or ironic way. So here, it might suggest that the evidence being presented as “key” might not actually be as strong or convincing as claimed.

3. Quotation within a quotation (depending on context): If the sentence is part of a larger quotation that already uses double quotes, the single quotes can show a quote within a quote. But since this sentence is isolated, this seems less likely unless there's a broader context.

So, in this case, the single quotes likely imply skepticism or quotation of someone else’s claim — as in, they're being called "key pieces of evidence", but the speaker may not entirely agree.

The next sections empasise this beginning using statements such as: "HDP is not accurate when applied to heterozygous individuals". Hence, one of the main points of contention is whether the individual used for assembly is heterozygous or homozygous.

Summary of Potentially Literary or Idiomatic Phrases:

Phrase	Type	Origin / Note
"In one fell swoop"	Direct literary allusion	Shakespeare, Macbeth
"Gold standard"	Idiomatic metaphor	20th c. journalism/lit
"Lines of evidence"	Technical phrase with rhetorical flair	Classical rhetoric
"All the evidence suggests..."	Forensic/narrative tone	Common in mystery/legal genres
"At this point, the single read can prove..."	Rhetorical flourish	Echoes legal or dramatic logic
"Another view is..."	Narrative pacing	Fiction/mystery-style argumentation

Patton Science paper and lack of evidence 

The papers by Patton et al. (2020) and Stammnitz et al. (2024) present opposing interpretations of the epidemiological trajectory of Devil Facial Tumor Disease (DFTD) in Tasmanian devils—Patton et al. argue that the disease is stabilizing into an endemic phase, while Stammnitz et al. refute this, finding no evidence for such a shift. This scientific dialogue mirrors the kind of tension seen in the Matters Arising exchange over the FM locus (fibromelanosis) in chickens, where initial claims about the genetic basis of the black-bone trait were later challenged through data reinterpretation and deeper comparative genomics. However, unlike a formal Matters Arising—where critique and rebuttal occur within the same journal—here the discourse spans across venues, illustrating a broader philosophical point: that scientific knowledge evolves not just through journal-specific dialogue, but through cross-platform, iterative analysis. Both cases reflect the self-correcting nature of science and the importance of maintaining open channels for debate beyond formalized comment formats.

Scientific Summaries

1. Patton et al. (2020) – Science

Title: A transmissible cancer shifts from emergence to endemism in Tasmanian devils

Summary: This study investigates the dynamics of Devil Facial Tumor Disease (DFTD), a transmissible cancer affecting Tasmanian devils. Utilizing an epidemiological phylodynamic approach, the researchers analyzed the disease's spread and adaptation over time. They found that in areas with lower devil densities, the disease's growth and transmission rates have slowed. This suggests that DFTD is transitioning from an emergent epidemic to an endemic state, implying a potential stabilization of the disease within the population.

2. Stammnitz et al. (2024) – Royal Society Open Science

Title: No evidence that a transmissible cancer has shifted from emergence to endemism in Tasmanian devils

Summary: Contrasting the findings of Patton et al., this study re-examines the status of DFTD in Tasmanian devils. Through comprehensive data analysis, the authors argue that there is no substantial evidence supporting the transition of DFTD from an emergent to an endemic state. They emphasize that the disease continues to pose significant threats to devil populations, and caution against assumptions of disease stabilization.

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Philosophical Perspective

Reconciling Contradictory Viewpoints

The divergence in conclusions between these two studies underscores the complexity of interpreting ecological and epidemiological data. Science often progresses through such debates, where differing methodologies, data sets, or analytical frameworks can lead to contrasting interpretations. This dynamic is not indicative of flaws but rather reflects the iterative nature of scientific inquiry, where hypotheses are continually tested and re-evaluated.

Role of Publication Venues

The venues of publication—Science and Royal Society Open Science—play a pivotal role in shaping the discourse. Science, being a high-impact journal, often publishes groundbreaking studies that can influence subsequent research directions. In contrast, Royal Society Open Science provides a platform for open peer review and encourages the publication of studies that may challenge prevailing narratives. This ecosystem allows for a balanced scientific dialogue, where initial findings can be scrutinized and refined through subsequent research.​

While Matters Arising articles are typically short critiques or follow-ups published in the same journal as the original work — often directly linked to the original article — the case here is philosophically and procedurally different:

• Same Topic, Different Journals: The original claim (DFTD shifting to endemism) was published in Science — a high-profile venue. The counterargument was published years later in Royal Society Open Science, an open-access journal that encourages broader debate and methodological transparency.

• Independent Platforms, Independent Framing: In a Matters Arising format, the critical article is tethered to the original and usually appears soon after. Here, Stammnitz et al. authored a full-length paper that challenges the entire framing of the earlier study, rather than submitting a formal comment to Science. This affords them greater space, nuance, and independence to re-analyze data and reframe conclusions.

• Philosophical Implication: This approach emphasizes that scientific truth does not reside within journals, but in rigorous process and reproducibility. It also reflects how open-access ecosystems like Royal Society Open Science serve as vital venues for long-form re-evaluation, even of claims made in elite journals.

This kind of exchange reinforces that science thrives on open, distributed critique, not just gated formats like Matters Arising. Both papers, by existing side by side in the literature, allow the community to weigh evidence, reproduce analyses, and refine our understanding — the essence of robust, evolving knowledge.