Optimization of DNA extraction and library preparation for metagenomic analysis of giant panda in Malaysia.

The objective of this study is to determine the expected minimum number of sequence reads read needed to achieve full coverage of the gut microbial species of the giant panda metagenome, firstly by analyzing 5 different mammalian metagenomes, namely that of the horse, coyote, whitetail deer, humpback whale and the bottlenose dolphin as reference; based on an approximate of 1 000 000 sequence read estimation. After rarefaction analysis using MG-RAST Version 3.0 analysis pipeline, an average value of 775 075 reads, which is close to expected 1 000 000 reads, is deduced. Next, the fecal matter of the giant panda is sampled at Zoo Negara, and metagenomic DNA extraction is performed to recover four final samples of raw metagenomic DNA, namely Panda A, Panda B, Panda C and Panda D. These four samples are then subjected to qualification and quantification analysis; where the results show that the samples are still viable and sufficient in yield to be used for library preparation, despite slight degradation and contamination during the extraction process. However, only Panda A and Panda B are selected for library preparation through six phase namely; DNA fragmentation, end repair and size selection, adenylate 3' ends, adapter ligation, library validation, as well as library normalization and pooling. Panda C and Panda D are kept as reserves. After library preparation, the pooled samples are sequenced using the Illumina™ MiSeq® next generation sequencer. The results of this research serve as a foundation for further studies of the giant panda metagenome.