Strategies for complete plastid genome sequencing

Abstract

Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.

Practical considerations for plant phylogenomics

“…Organellar genomes make up a large component of total genomic DNA, with cpDNA ranging from <0.3% in Picea abies (L.) H. Karst. needles to 37% in Asclepias syriaca L. leaves (Twyford and Ness, ) and mitochondrial DNA abundance 5–10% that of cpDNA (Bock et al., ). Although genome size in flowering plants varies from 63.6 Mbp in Genlisea aurea A. St.‐Hil.…”

“…Organellar genomes make up a large component of total genomic DNA, with cpDNA ranging from <0.3% in Picea abies (L.) H. Karst. needles to 37% in Asclepias syriaca L. leaves (Twyford and Ness, 2016) and mitochondrial DNA abundance 5-10% that of cpDNA (Bock et al, 2014). correlation between genome size and percent total organellar DNA, suggesting that genome skimming is a viable option for obtaining organellar genomes across many taxa (Bakker et al, 2016;Twyford and Ness, 2016).…”

“…needles to 37% in Asclepias syriaca L. leaves (Twyford and Ness, 2016) and mitochondrial DNA abundance 5-10% that of cpDNA (Bock et al, 2014). correlation between genome size and percent total organellar DNA, suggesting that genome skimming is a viable option for obtaining organellar genomes across many taxa (Bakker et al, 2016;Twyford and Ness, 2016). Because of their relative abundance, (usual) structural simplicity, and historical significance in systematics, chloroplast genomes have become a primary target of genome skimming projects.…”

Herbarium genomics: skimming and plastomics from archival specimens

“…Twyford and Ness (2016) summarize current strategies for complete plastome sequencing and conclude that most of them include genome skimming and omit targeted plastome-enrichment or the isolation of organelles before DNA extraction. As the authors indicate basically two approaches to plastome assembly can be distinguished: mapping and assembling reads to a reference genome, or de novo assembly.…”

“…Sequencing from libraries made from un-enriched, total genomic extract means that nuclear DNA will represent most of the DNA. For a genome size of say 440 Mb (Mimulus guttatus) the ratio of plastid to nuclear genome coverage is approximately 67 : 1, which implies that 3.1% of all reads are derived from the plastid (Twyford and Ness 2016). If 500 Mb of nucleotides were to be sequenced (which is a small amount given current high-output sequencing and Standley 2013), see Figure 1; one would expect co-linearity of assembly and reference plastome in case of conspecifics or closely-related target and reference species.…”