The most important advantages of this cloning-independent protein inhibitors approach are the avoidance of cloning bias and the bias introduced by PCR amplification. To best of our knowledge, this study was the first to apply a random sample pyrosequencing approach to analyze the metagenome of the pygmy loris to better understand how microbiomes relate to NHPs ecological and evolutionary diversity. Materials and Methods Fecal Sample Collection Fresh fecal samples from two pygmy loris were collected from the Daweishan Nature Reserve of Pingbian, Yunnan Province, China, with the permission of the authorities of the Daweishan Nature Reserve of Pingbian. We tracked the two pygmy loris until they defecated; the fecal samples were immediately collected aseptically.
The fresh fecal samples were transported to the laboratory on dry ice within 24 hours of collection, and then stored at ?80��C until DNA extraction. We brought no toxic substance that would have adverse effects on the biotic community to minimize disturbance in the animal habitats. The research complied with the protocols established by the China Wildlife Conservation Association and adhered to the American Society of Primatologists (ASP) Principles for the Ethical Treatment of Non-Human Primates as well as the legal requirements of China. DNA Extraction and Shotgun Pyrosequencing Genomic DNA were extracted from the fecal samples with the QIAamp DNA stool mini kit (Qiagen, Valencia, CA, USA) following the protocol provided by the supplier (0.25 g of each fecal sample).
The quality and quantity of the DNA were determined with a nanodrop (ND-1000) spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) through agarose gel electrophoresis. The DNA samples from the two pygmy loris were pooled on an equimolar basis. DNA samples were stored frozen (?20��C) until use. A total of 500 ng of pooled DNA was subjected to library preparation and shotgun pyrosequencing using the Roche 454 GS FLX Titanium System (Roche, Basel, Switzerland). The obtained reads were uploaded to a Metagenome Rapid Annotation Using Subsystem Technology (MG-RAST)  under the name WFH_Metagenome and were assigned the Metagenome ID: 4476304.3. The MG-RAST v.3.0 online server quality control pipeline was utilized to remove reads of short length and poor quality before annotation and the analysis of metagenomic data .
The pipeline parameters were kept at default settings. The raw sequencing reads were submitted to the Joint Genome Institute’s IMG/M-ER Dacomitinib annotation pipeline . Bioinformatics and Statistical Analysis Comparative metagenomic analysis was performed with both the MG-RAST and IMG/M pipelines. The metagenomic runs from the pygmy loris data were compared with the current publicly available gut metagenomes in each of the two databases.