宏觀基因體學(Metagenomics)是直接去研究環境中微生物DNA的研究。用下世代定序平台，將這些微生物DNA序列定序，將序列對上已知物種的DNA資料庫，以得知微生物的物種。下世代定序平台有Illumina和454，比以往Sanger定序能以更低的成本產生更多的序列。454定序平台可以產生序列比較長( 700-800 base pair )，但是成本較為昂貴，隨著技術的改善Illumina paired-end read定序可以達到600 base pair( 2x300 base pair )，並且以更低的成本產生更多的序列。所以使用Illumina定序平台來進行宏觀基因體學的研究越來越熱門。以往研究都是以454 single-end read定序為主(序列長度較長)，但目前還沒有研究評估物種鑑定工具對於paired-end read的準確度。本研究會以模擬的paired-end read資料為主，分別產生兩種資料: (1) large (amplicon size 大於600 base pair, i.e., non-overlapping) and (2) small (amplicon size 400-500 base pair, i.e., overlapping)。然後分別去用Blast, RDP, SOAP2和Bowtie2進行物種鑑定，計算各個物種鑑定工具的準確率。在兩種模擬資料中，使用RDP是最準確的。本研究首次探討對於paired-end read資料，RDP為較佳之物種鑑定工具。

Metagenomics is the study of microbial DNAs from environmental samples. Next-generation sequencing (NGS) can produce much more reads with a lower cost than Sanger sequencing. There are two common NGS platforms, i.e., Illumina and 454. Although 454 platform offers read lengths up to 800 bp, Illumina platform produces more reads with a lower cost than 454 and offers read lengths up to 600 bp (2x300 bp). So many researchers prefer Illumina in metagenomic studies. In previous studies, no research explored the classification accuracy using Illumina paired-end reads. In our study, we evaluated classification accuracy of four popular tools, i.e., Bowtie2, SOAP2, RDP and Blast. We used SILVA 16S rDNA database for simulating Illumina reads (2x300 bp) with two 16S primer sets: (1) larger dataset containing non-overlapping paired-end reads and the amplicon size was over 600bp; (2) small dataset containing overlapping paired-end reads and the amplicon size was between 400-500bp. We compared classification accuracy of Bowtie2, SOAP2, Blast and RDP. The results showed that RDP was the most accurate. Thus, we suggested that RDP was a better tool for species classification with Illumina PE data.

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