Software Choice and Sequencing Coverage Can Impact Plastid Genome Assembly–A Case Study in the Narrow Endemic Calligonum bakuense

Abstract

Most plastid genome sequences are assembled from short-read whole-genome sequencing data, yet the impact that sequencing coverage and the choice of assembly software can have on the accuracy of the resulting assemblies is poorly understood. In this study, we test the impact of both factors on plastid genome assembly in the threatened and rare endemic shrub Calligonum bakuense. We aim to characterize the differences across plastid genome assemblies generated by different assembly software tools and levels of sequencing coverage and to determine if these differences are large enough to affect the phylogenetic position inferred for C. bakuense compared to congeners. Four assembly software tools (FastPlast, GetOrganelle, IOGA, and NOVOPlasty) and seven levels of sequencing coverage across the plastid genome (original sequencing depth, 2,000x, 1,000x, 500x, 250x, 100x, and 50x) are compared in our analyses. The resulting assemblies are evaluated with regard to reproducibility, contig number, gene complement, inverted repeat length, and computation time; the impact of sequence differences on phylogenetic reconstruction is assessed. Our results show that software choice can have a considerable impact on the accuracy and reproducibility of plastid genome assembly and that GetOrganelle produces the most consistent assemblies for C. bakuense. Moreover, we demonstrate that a sequencing coverage between 500x and 100x can reduce both the sequence variability across assembly contigs and computation time. When comparing the most reliable plastid genome assemblies of C. bakuense, a sequence difference in only three nucleotide positions is detected, which is less than the difference potentially introduced through software choice.

Genomic Underground: Unraveling NUMTs in Mole Voles

“…It is probable that this duplication occurred because of the use of short reads and the De Bruin graph for sequence assembly 47 . Although GetOrganelle has demonstrated the highest accuracy among all published plastid assembly pipelines in benchmarks [48][49][50] , this observation underscores the importance of the molecular validation of in silico results.…”

Depth and evenness of sequencing coverage are associated with assembly quality, genome structure, and choice of sequencing platform in archived plastid genomes

“…Similarly, Freudenthal et al [30] used sequencing coverage as part of a special metric to evaluate the accuracy of plastid genome assemblies and found that this metric differed strongly between different plastome assembly software. Giorgashvili et al [31] tested the opposite association and reported that plastid genome assembly software tools generated different contigs depending on the levels of sequencing coverage. Hence, software choice most likely has a considerable impact on the results of plastome assembly, including its sequencing coverage [101], even if sequencing evenness may not be affected at a significant level.…”

“…However, the average plastome-wide sequencing depth is the only assembly quality metric that is somewhat frequently applied in contemporary plastid genomics. Twyford and Ness [29] discussed its use in identifying the overall adequacy of sequencing coverage in plastid genomics, while studies such as Freudenthal et al [30] and Giorgashvili et al [31] employed it to determine the accuracy of de novo plastid genome assemblies. Except for Gu et al [6], sequencing evenness has barely been used as a quality metric for plastid genome assemblies, even though the precise distribution of sequencing depth can help identify genome regions of inadequate coverage.…”

“…The post-processing and scaffolding of contigs are known to introduce gaps and ambiguous nucleotides into genome assemblies [12,40], which likely generates a distinct variation in sequencing depth or evenness across the assembly. This variation may be further pronounced by the quadripartite genome structure of the plastid genomes of most land plants [31]. Researchers need to take this variation into consideration when employing sequencing depth or sequencing evenness as assembly quality indicators.…”

Chlomito: a novel tool for precise elimination of organelle genome contamination in nuclear genome assemblies

“…Meanwhile, the SDR refers to the ratio of each coting’s sequencing depth to the average sequencing depth of the organelle genome. Given that organelle genomes exist in multiple copies within a cell, they typically exhibit higher sequencing depths than nuclear genome(Sanita Lima et al, 2016;Wang et al, 2018;Li et al, 2021;Giorgashvili et al, 2022;Zhou et al, 2023a). Therefore, a contig with a high sequencing depth ratio, similar to the average of the organelle genome, is more likely to be a part of the organelle genome.…”