# Variation of and associations with the depth and evenness of sequencing coverage in archived plastid genomes

## TL;DR

This study examines how sequencing depth and evenness vary in archived plastid genomes and how these factors relate to genome structure and assembly quality.

## Contribution

The study provides new insights into how sequencing coverage metrics vary across plastid genome partitions and relate to assembly quality and methodological factors.

## Key findings

- Significant differences in sequencing depth exist across genome partitions and coding vs. non-coding regions.
- Sequencing evenness correlates with ambiguous nucleotides and varies between sequencing platforms.
- Normalizing input sequence data before assembly may improve plastome assembly accuracy.

## Abstract

Depth and evenness of sequencing coverage are considered potential indicators of genome assembly quality. In plastid genomics, where new data generation has outpaced the development of assembly quality indicators, these coverage metrics could offer insights into the quality of plastomes of different sizes, structures, or taxonomic origins. However, the variation of sequencing depth and evenness among archived plastid genomes, their variability between genome partitions, and any association with methodological factors have yet to be evaluated. This study explores the variation of sequencing depth and evenness across a sample of publicly accessible plastid genomes in relation to their genome structure, assembly quality, and methodological provenance using uni- and multivariate statistical analyses. We also evaluate whether sequencing evenness in plastid genomes is biased by phylogenetic signal and assembly software choice, and whether more uniformly distributed input sequence data improves plastome assembly quality. Our results indicate significant differences in sequencing depth across the four structural partitions and between the coding and non-coding regions of plastid genomes, a significant correlation between sequencing evenness and the number of ambiguous nucleotides, and a significant difference in sequencing evenness between sequencing platforms. However, we also find that different covariates representing additional, lesser explored factors often show a similar, if not greater, explanatory power for the coverage variation. No indications of phylogenetic or software choice bias on sequencing evenness and only weak indications of phylogenetic bias among the assembly quality metrics are detected, suggesting that our study results represent genuine patterns. We also find that normalizing the distribution of the input sequence data before plastome assembly may improve assembly accuracy. Taken together, these findings highlight that many public plastid genomes derive from sequence data with highly variable depth and evenness, and that this variation is influenced, at least partially, by genome structure as well as methodological factors.