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HybSeq Workshop Case Studies

1. Fresh samples, no genomic resources, phylogenetics

You would like to reconstruct the phylogeny of a medium-sized genus of rosids. Due the industrious collecting of your colleagues, you have at least two accessions each of all 47 species growing in your research greenhouse. However, due to the ugliness of the plants and their total lack of commercial value, no genomic resources exist within the relevant family (although published draft genomes exist for two taxa in the same order).

2. Fresh samples, transcriptome resources, functional genes

You are interested in co-evolution of inflorescence size in your pet genus with jaw width in a clade of florivorous Rodents of Unusual Size; as part of this investigation, you would like to test for selection in candidate genes involved in floral development. You have therefore joined a large collaborative project investigating directional selection in 108 MADS-box genes in a clade of angiosperms containing two orders, six families, 76 genera (including yours), and 2,382 species. After missing a critical task-assignment meeting, you find yourself tasked with designing a HybSeq study to capture the genes. You have access to one transcriptome assembly per family for five families, and three transcriptomes for the sixth family, to aid you in developing your markers.

3. Mix of herbarium and fresh samples, low-coverage genome, functional genes

In a welcome stoke of good fortune, you have been awarded a seed grant to investigate pseudogenization and copy-number variation in 112 putative defense genes in a 4-species complex of a commercially-important horticultural plant with beautiful but disease-prone flowers. You suspect that a domestication bottleneck has led to disease-prone plants in cultivation. Available material includes living collections of 27 modern named varieties, and historical specimens in herbaria from 18 wild populations, some of which you suspect may have been sources of material brought into cultivation in the 19th century. You have a low-coverage genome assembly for one of the modern varieties as well as a published draft genome from the sister family.

4. Mix of herbarium and fresh samples, no genomic resources, phylogenetics

Due to an extremely ill-advised bet, your general well-being heavily depends on reconstructing an accurate phylogeny of two sister ascomycete genera containing 142 species. Although you have access to silica-dried material for 27 species, the remaining species are available only in herbaria due to a combination of extinction and a prolonged civil war making fieldwork impractical. For those 115 species, the most recently collected specimen ranges from 110 to 7 years old. Four species exist only as type specimens in a small regional herbarium, so, in the words of the curator, “this had better work.” No genomic resources exist within the family, but one low-coverage published draft genome exists in the sister family.  

Planning your HybSeq project

What you need and what it costs.

Outline a strategy for developing HybSeq baits appropriate for your study.

Be sure to include

  • A proposed method for marker development, including any required sequencing
  • Sources of phylogenetically-appropriate bait sequences
  • The estimated total cost of bait development.

Roughly estimate the cost of the lab work required to carry out the project.

At this stage in the workshop it will only be a rough estimate. Estimate the number of samples and cost of consumable required (including kits) for the following:

  • DNA extraction

  • Library preparation

  • Hybridization

  • Sequencing

Add together your cost estimates: what is the total cost?

Then suppose you only have one-quarter of these funds available. How would you structure a pilot study that would (hopefully) produce enough results to justify a request for full funding?