Long-read assemblies reveal structural diversity in genomes of organelles - an example with Acacia pycnantha

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Although organelle genomes are typically represented as single, static, circular molecules, there is evidence that the chloroplast genome exists in two structural haplotypes and that the mitochondrial genome can display multiple circular, linear or branching forms. We sequenced and assembled chloroplast and mitochondrial genomes of the Golden Wattle, Acacia pycnantha, using long reads, iterative baiting to extract organelle-only reads, and several assembly algorithms to explore genomic structure. Using a de novo assembly approach agnostic to previous hypotheses about structure, we found different assemblies revealed contrasting arrangements of genomic segments; a hypothesis supported by mapped reads spanning alternate paths.

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    Reviewer 1. Aboozar Soorni

    Have any claims of performance been sufficiently tested and compared to other commonly-used packages? no Additional Comments Are there (ideally real world) examples demonstrating use of the software? No Additional Comments Is automated testing used or are there manual steps described so that the functionality of the software can be verified? No

    Recommendation: Accept

    **Reviewer 2. Weiwen Wang ** This manuscript is easy to read, with good writing and details of methods. By comparing the long-read, short-read and hybrid assembly, this manuscript found out the best approach to assemble plastid and mitochondrial genome. Additionally, authors considered the multiple structures of plastid and mitochondrial genome, and providing a new and carefully designed method to assemble and assess the complex mitochondrial genome. While this manuscript represents a solid work and it was interesting to read it, I have some minor concerns which should be fixed to improve the quality of the manuscript. Line 240-247: Maybe it could be easier to understand if authors can number each contig. For example, “The assembly graph suggests the typical quadripartite structure of a LSC (contig 1-7) as the larger circle in the graph…...”. In some figures, authors numbered the contigs, but in some did not. Also, in the figure 2, why does the SSC region also have almost 2x coverage (1.88x)? Line 253-258: In the figure 3, it is three contigs (92 kb, 38 kb and 5kb) rather than two contigs (81 kb and 92 kb) that this manuscript described. I guess authors put the wrong figure? Line 283-285: It is a smart method to clearly show the assembly details. Line 385-387: Does it mean that the black segment (edge 11) in Figure 12 is consisted of two highly similar (or the same) regions? Have authors tried to do a simple BLAST to confirm this?