Developing Novel Amplicon Sequencing Assays From Scat For Diet Analysis of the pacific pocket mouse (perognathus longimembris pacificus) Active

Amplicon sequencing of scat using “Next Generation” sequencing techniques can revolutionize this field of study.  Amplicon sequencing methods use a set of genetic barcodes to amplify all contributing components in a DNA sample (in this case a scat) at one time, and next generation sequencing platforms can analyze thousands to hundreds of thousands of individuals per run.  These technologies will allow detectability and quantification of diet components at levels not achieved with microscopy, with limited or no disturbance to individual animals.  While such sequencing techniques have been developed and successfully employed frequently with carnivores, herbivorous animals may present additional challenges due to chemical inhibitors present in plants, and fewer genetic barcoding resources for local plant communities. Therefore, there are very few examples of herbivorous pcr-based diet studies currently found in the scientific literature.   The US Geological Survey has been studying the ecology of the pacific pocket mouse (Perognathus longimembris pacificus; PPM) since 2007 to build a knowledge base for sound management and restoration. Understanding PPM diet can help provide information on habitat requirements and to develop habitat restoration goals consistent with supporting PPM populations. This study to determine diet from fecal samples was initiated in 2014 with an initial goal of determining whether a metabarcoding approach would be feasible in PPM.

Objectives:

1. Refine the PCR amplification protocol to focus specifically on the ITS2 gene region to improve recovery and length of informative sequences (thus improve assignments);
2.  Supplement and refine the ITS2 database to restrict to field observed plant species (thus reducing false positive plant identifications);
3.  Determine sensitivity and accuracy through further analysis of “mock samples” comprised of known plant DNA in proportions relevant to field observations and additional fecal samples of captive bred PPM;
4. Prepare a comprehensive report of results.

References:

Iwanowicz, D.D., A. G. Vandergast, R. S. Cornman, C. R. Adams, J. R. Kohn, R. N. Fisher, and C.S. Brehme, 2016, Metabarcoding of fecal samples to determine herbivore diets: A case study of the endangered pacific pocket mouse. PLos ONE 11(11): e0165366. Doi:10.1371/journal.pone.0165366

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