GCAT-SEEKquence
The Genome Consortium for Active Teaching
NextGen Sequencing Group
NextGen Sequencing Request Form
Complete fields below, save file with your last name at the beginning of
the filename (e.g. newman-GCAT-SEEK Sequence request form.pdf) and
email to Vincent Buonaccorsi <BUONACCORSI@juniata.edu>
A. Contact Information
1. Name:
A. Malcolm Campbell (lead PI) and Michael Dorcas
2. Department: Biology
3. Institution: Davidson College
4. Phone Number: 704-894-2692
5. Email Address macampbell@davidson.edu, midorcas@davidson.edu,
B. Project Information
1. Title: Migration of green tree frog into historically more temperate regions. (Hyla cinerea)
2. Category: transcriptomes (Wang et al., 2009)
3. Total amount of sequence requested: We will need two to three lanes.
4. Preferred technology: Illumina
5. Do you have funds for a partial run next Spring? no
C. Describe the background, hypotheses and specific aims (500 words max)
The green tree frog (Hyla cinerea) historically was limited to the Coastal Plain and lower Piedmont of the
Carolinas However, within the last 10 years, this species has expanded its range to encompass much of the
Piedmont (unpublished data). We want to understand whether the transcriptome is changing by comparing
transcriptomes of green tree frogs isolated in the upper Piedmont to those isolated from the Coastal Plain.
We would also like to compare the green tree frog with the gray tree frog (Hyla chrysoscelis), a species that
has always lived throughout both North and South Carolina. What has changed? Is climate change the cause
and the transcriptomes are unchanged (Stuart et al., 2004; Araújo, et al., 2006; Lips, et al., 2008)? Or is the
green tree frog evolving and producing a different transcriptome that permits it to survive the colder
conditions?
It is worth noting that the only anuran genomic data publicly available is the west African X. tropicalis
(http://faculty.virginia.edu/xtropicalis/). Searching NCBI database revealed very few entries for any anuran
genes or proteins. To our knowledge, this would be the first genomic-level project on any anuran from the
Americas. We hope to use this information to provide a good starting place to sequence complete genomes
in the future.
D. Describe the methods [sample prep, calculation of amount of sequence required, analysis plan]
One lane would give 37.5GBp of PE sequence. We will need around 30X coverage for assembly. This
would be 1.5% of 4GBp * 30 which works out to about 2GBp per transcriptome. We will barcode and get 15
samples analyzed in the same lane. We will sample three different sources of RNA: 1) gray tree frog endemic
to Piedmont of NC; 2) green tree frog from endemic Coastal Plain; 3) green tree frog from the Piedmont of
NC. We will sample tadpoles and metamorphosing young frogs at five different developmental stages to
produce 15 samples of mRNA. We will prepare the 15 samples in triplicate from three different collections
of biological material to provide greater population diversity and redundancy for assembly.
We are searching for two teaching postdocs funded by Davidson College to lead this interdisciplinary
effort. The bioinformatics postdoc will work with undergraduates to assemble the reads and annotate the
deduced transcripts/genes. We would assemble the reads using NextGENe, and count using NextGENe or
Galaxy programs. The conservation biologist will head up all field work in collaboration with undergraduate
researchers at Davidson College.
E. Describe the role and number of undergraduates involved in the project, and how they would benefit.
Undergraduates from the Dorcas lab will be responsible for collecting the biological samples and
documenting their age and geographical location. Undergraduates from the Campbell lab will be responsible for
isolating the RNA and keeping track of each biological source. Dorcas and Campbell will mentor the two teaching
postdocs as they work with undergraduates. The postdocs will work collaboratively to assemble and annotate
the transcriptome in conjunction with all the students enrolled in Laboratory Methods in Genomics which is
required for the Genomics Concentration. In addition, the conservation biology postdoc will work with
undergraduates to survey the region to document the distribution of gray and green tree frogs. This team of
students and postdocs will integrate the animal distribution data with historical weather records and species
distribution records to better understand the migration of the green tree frog.
The conservation biology team and the bioinformatics/genomics team will work collaboratively to determine
if climate and/or genomic changes best explain the new migration of green tree frogs. Dorcas and Campbell
each typically mentor about 8 undergraduates a year. In addition, the postdocs will be teaching courses related
to this collaborative project. Over the three-year period, we estimate approximately 60 undergraduates will be
directly involved in this transcriptome project.
F. I agree to administer the GCAT-SEEK pre- and post-activity assessment test for students and to complete the
faculty post-utilization survey. _X yes, ____ no
G. Describe any other broader impact or intellectual merit considerations.
Xenopus tropicalis is the only anuran that has been sequenced and this would be the first North American
anuran sequenced. Since amphibians are suffering a radical decline in numbers globally, it would be good to
know the genomes of many species to help us understand what might permit one species to survive but not
another one.
H. References
Araújo, M. B., W. Thuiller, and R. G. Pearson. 2006. Climate warming and the decline of amphibians and reptiles
in Europe. Journal of Biogeography. Vol. 33: 1712–1728.
Lips, Karen R., Jay Diffendorfer, Joseph R. Mendelson III, Michael W. Sears. 2008. Riding the Wave: Reconciling
the Roles of Disease and Climate Change in Amphibian Declines. PLoS Biology. Volume 6(3): 441 – 454.
Stuart, Simon N., Janice S. Chanson, Neil A. Cox, Bruce E. Young, Ana S. L. Rodrigues, Debra L. Fischman, Robert
W. Waller. 2004. Status and Trends of Amphibian Declines and Extinctions Worldwide. Science. Vol. 306: 1783 –
1786.
Wang, Zhong, Mark Gerstein and Michael Snyder. 2009. RNA-Seq: a revolutionary tool for transcriptomics.
Nature Reviews. Vol. 10: 57 – 63.