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.