Strategies for reducing per‐sample costs in target capture sequencing for phylogenomics and population genomics in plants

Abstract

Citation: Hale, H., E. M. Gardner, J. Viruel, L. Pokorny, and M. G. Johnson. 2020. Strategies for reducing per-sample costs in target capture sequencing for phylogenomics and population genomics in plants. Applications in Plant Sciences 8(4): e11337.The reduced cost of high-throughput sequencing and the development of gene sets with wide phylogenetic applicability has led to the rise of sequence capture methods as a plausible platform for both phylogenomics and population genomics in plants. An important consideration in large targeted sequencing projects is the per-sample cost, which can be inflated when using off-the-shelf kits or reagents not purchased in bulk. Here, we discuss methods to reduce per-sample costs in high-throughput targeted sequencing projects. We review the minimal equipment and consumable requirements for targeted sequencing while comparing several alternatives to reduce bulk costs in DNA extraction, library preparation, target enrichment, and sequencing. We consider how each of the workflow alterations may be affected by DNA quality (e.g., fresh vs. herbarium tissue), genome size, and the phylogenetic scale of the project. We provide a cost calculator for researchers considering targeted sequencing to use when designing projects, and identify challenges for future development of low-cost sequencing in non-model plant systems. KEY WORDS enzymatic fragmentation; herbariomics; high-throughput workflow implementation; Hyb-Seq; low-cost sequence capture; pooling and multiplexing strategies. Applications in Plant Sciences 2020 8(4): e11337 Hale et al.-Low-cost Hyb-Seq • 2 of 10

Repeated parallel losses of inflexed stamens in Moraceae: phylogenomics and generic revision of the tribe Moreae and the reinstatement of the tribe Olmedieae (Moraceae)

“…We sampled 56 out of 66 species (and all genera) in Moreae as well as select outgroup taxa from Artocarpeae using DNA from leaf tissue preserved on silica gel or-in most cases-samples from herbarium specimens (Table S1). In either case, DNA was extracted using a modified CTAB method, usually with increased incubation times to maximize yield from herbarium tissue (Doyle & Doyle, 1987;Hale & al., 2020). Samples were quantified using a Qubit fluorometer (Invitrogen, Life Technologies, California, USA), and herbarium samples were run on a gel to test for degradation.…”

Niche evolution of the Neotropical tree genusOtobain the context of global biogeography of the nutmeg family, Myristicaceae

“…Target enrichment was carried out using the MyBaits Angiosperms353 universal probe set (Däicel Arbor Biosciences, Ann Arbor, MI; (Johnson et al, 2019)). Target enrichment followed the modifications to the manufacturer's protocol outlined in (Hale et al, 2020); i.e., pool of 20-24 samples and RNA baits diluted to ¼ concentration). Unenriched DNA library was added to the cleaned, target enriched pool to increase the amount of off-target, chloroplast fragments in the sequencing library.…”

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“…were prepared using the NEBNext Ultra II DNA Prep Kit following the published protocol for this kit (New England Biolabs, Ipswitch, MA, USA), and quantified using an Agilent Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Santa Clara, CA, USA). Libraries were pooled at equimolar concentrations into two pools of six prior to probe hybridization following the Arbor Biosciences myBaits protocol v 3.0.1 andHale et al (2020). The hybridized samples were subsequently pooled at equimolar ratios and sequenced at the Texas Tech Center for Biotechnology and Genomics using a MiSeq with the Micro chemistry and 150 bp paired-end reads (Illumina, Inc., San Diego, CA, USA).…”