A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes

Abstract

• Premise of the study: We explored a targeted enrichment strategy to facilitate rapid and low-cost next-generation sequencing (NGS) of numerous complete plastid genomes from across the phylogenetic breadth of angiosperms.• Methods and Results: A custom RNA probe set including the complete sequences of 22 previously sequenced eudicot plastomes was designed to facilitate hybridization-based targeted enrichment of eudicot plastid genomes. Using this probe set and an Agilent SureSelect targeted enrichment kit, we conducted an enrichment experiment including 24 angiosperms (22 eudicots, two monocots), which were subsequently sequenced on a single lane of the Illumina GAIIx with single-end, 100-bp reads. This approach yielded nearly complete to complete plastid genomes with exceptionally high coverage (mean coverage: 717×), even for the two monocots.• Conclusions: Our enrichment experiment was highly successful even though many aspects of the capture process employed were suboptimal. Hence, significant improvements to this methodology are feasible. With this general approach and probe set, it should be possible to sequence more than 300 essentially complete plastid genomes in a single Illumina GAIIx lane (achieving ∼50× mean coverage). However, given the complications of pooling numerous samples for multiplex sequencing and the limited number of barcodes (e.g., 96) available in commercial kits, we recommend 96 samples as a current practical maximum for multiplex plastome sequencing. This high-throughput approach should facilitate large-scale plastid genome sequencing at any level of phylogenetic diversity in angiosperms.

A Plastid Phylogeny of the Cosmopolitan Fern Family Cystopteridaceae (Polypodiopsida)

“…These tools will no doubt soon be available ( Leaché et al, 2014 ), as will increasingly longer read lengths of reduced representation libraries. These considerations notwithstanding, we feel strongly that pairing herbarium collections with GBS and other increasingly accessible genomic workfl ows ( Straub et al, 2012 ;Stull et al, 2013 ;Weitemier et al, 2014 ) should be a top priority in plant systematics. Besides allowing for rapid and economical sampling of large groups, next-generation sampling allows specimen selection to be performed in collaboration with group experts.…”

“…This type of sampling is, however, still viewed by most as a way to supplement an otherwise fi eld-collected data set. Although studies utilizing genomic data sets obtained from herbarium specimens are emerging, most involve the recovery of high-copy organelle and/or rDNA cistron regions ( Straub et al, 2012 ;Stull et al, 2013 ;Besnard et al, 2014 ;Ripma et al, 2014 ), or are focused on adaptation within a single species ( Vandepitte et al, 2014 ) or genome assembly of a single individual ( Staats et al, 2013 ). Indeed, we are unaware of a study that has performed species delimitation or phylogeny reconstruction using a genome-wide data set obtained exclusively (or even largely) from herbarium material.…”

“…Sampling exclusively from herbarium material would allow robust taxonomic and geographic sampling to be achieved rapidly, and if this sampling were performed under the guidance of expert taxonomists it would also ensure the strongest link between taxonomy and DNA. Sample sets obtained through this strategy, what we term "next-generation sampling ," could then be subjected to next-generation genotyping and sequencing techniques, as these workfl ows are presumably applicable to the sheared DNAs obtained from museum specimens ( Nachman, 2013 ;Stull et al, 2013 ;Burrell et al, 2015 ). These rich data was used for tag alignment and subsequent SNP calling.…”

The quest to resolve recent radiations: Plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae)

“…Most ancient DNA is generally fragmented to sizes less than 100 bp, and so the PCR approach may preclude successful results from poorly preserved samples (Knapp and Hofreiter, 2010). Other enrichment approaches, such as hybridization-based sequence capture (Stull et al, 2013), may circumvent this to some degree, but targeting short sequences is still challenging (Ávila-Arcos et al, 2015).…”

Phylogenomics of Andropogoneae (Panicoideae: Poaceae) of Mainland Southeast Asia

“…Advances in high throughput DNA sequencing have made recovery of chloroplast gene sets, and whole chloroplast genomes, increasingly easier (Steele et al 2012;Stull et al 2013). A number of studies have taken advantage of this technology, using either chloroplast protein-coding genes (e.g.…”