A multi–step comparison of short–read full plastome sequence assembly methods in grasses

Abstract

Technological advances have allowed phylogenomic studies of plants, such as full chloroplast genome (plastome) analysis, to become increasingly popular and economically feasible. Although next–generation short–read sequencing allows for full plastomes to be sequenced relatively rapidly, it requires additional attention using software to assemble these reads into comprehensive sequences. Here we compare the use of three de novo assemblers combined with three contig assembly methods. Seven plastome sequences were analyzed. Three of these were Sanger–sequenced. The other four were assembled from short, single–end read files generated from next–generation libraries. These plastomes represented a total of six grass species (Poaceae), one of which was sequenced in duplicate by the two methods to allow direct comparisons for accuracy. Enumeration of missing sequence and ambiguities allowed for assessments of completeness and efficiency. All methods that used de Bruijn–based de novo assemblers were shown to produce assemblies comparable to the Sanger–sequenced plastomes but were not equally efficient. Contig assembly methods that utilized automatable and repeatable processes were generally more efficient and advantageous when applied to larger scale projects with many full plastomes. However, contig assembly methods that were less automatable and required more manual attention did show utility in determining plastomes with lower read depth that were not able to be assembled when automatable procedures were implemented. Although the methods here were used exclusively to generate grass plastomes, these could be applied to other taxonomic groups if previously sequenced plastomes were available. In addition to comparing sequencing methods, a supplemental guide for short–read plastome assembly and applicable scripts were generated for this study.

The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): Comparative analyses and molecular dating

“…The comparison of the chloroplast genomes of Spartina maritima and the recently sequenced Chloridoideae Neyraudia reynaudiana (Wysocki et al, 2014) contributed to fill an important gap in the knowledge of plastid genome evolution in this group and revealed variable regions of possible taxonomic and phylogenetic utility in this large subfamily. We used some of these markers to bring new insights on the evolutionary history and divergence times of the Spartina lineages, of particular interest in the context of the prominent reticulate history of this group.…”

“…1600 species in 31 genera and exhibit a worldwide distribution (Peterson et al, 2007;Soreng et al, 2015), only one Chloridoideae plastome has been sequenced: Neyraudia reynaudiana (Wysocki et al, 2014), which belongs to another tribe (Triraphideae, Soreng et al, 2015). This contrasts with other Poaceae subfamilies such as the Pooideae or Bambusoideae for which twelve and eight plastomes are available respectively (NCBI Organelle Genome Resources, www.ncbi.nlm.nih.gov/genomes/ accessed on 30…”

Resolving deep relationships of PACMAD grasses: a phylogenomic approach

“…Four genera within Danthonioideae were also included to represent the danthonioid lineage as well as a representative species of Aristidoideae, Aristida purpurea (Aristideae) . The only published plastome for a chloridoid species, Neyraudia reynaudiana , was also included in our analyses [29]. …”

“…The Velvet software package [34] was run iteratively following previously established methods [26] to assemble reads into contiguous sequences (contigs). Contigs were scaffolded using the anchored conserved region extension (ACRE) method [29]. Conserved regions were identified using a grass family-wide alignment of plastomes.…”

Complete chloroplast genomes of Saccharum spontaneum, Saccharum officinarum and Miscanthus floridulus (Panicoideae: Andropogoneae) reveal the plastid view on sugarcane origins

“…As a consequence of whole genome sequencing, plastome (plastid and mitochondrial) genomes are typically sequenced concomitantly with the nuclear genome (Bolger et al, 2014). In addition, recent advances in genome assembly algorithms have made the assembly of de-novo sequence fragments not only possible, but also relatively easy, as long as a closely related genome is available for use as a template (Wysocki et al, 2014). Many plant genome sequencing projects deposit their raw read data in the NCBI Sequence Read Archive (SRA) (http://ncbi.nlm.nih.gov/sra).…”