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Improved pipeline for reducing erroneous identification by 16S rRNA sequences using the Illumina MiSeq platform

  • Systems and Synthetic Microbiology and Bioinformatics
  • Published:
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Abstract

The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3′-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended method corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform.

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Authors and Affiliations

  1. ChunLab, Inc., Seoul National University, Seoul, 151-742, Republic of Korea

    Yoon-Seong Jeon, Jeongmin Lim, Jongsik Chun & Bong-Soo Kim

  2. Interdisciplinary Graduate Program in Bioinformatics, Seoul National University, Seoul, 151-742, Republic of Korea

    Yoon-Seong Jeon & Jongsik Chun

  3. School of Biological Sciences and Bioinformatics Institute, BIO-MAX, Seoul National University, Seoul, 151-742, Republic of Korea

    Sang-Cheol Park & Jongsik Chun

  4. Department of Life Sciences, Hallym University, Chuncheon, Gangwon-do, 200-702, Republic of Korea

    Bong-Soo Kim

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  1. Yoon-Seong Jeon
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Correspondence to Bong-Soo Kim.

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Jeon, YS., Park, SC., Lim, J. et al. Improved pipeline for reducing erroneous identification by 16S rRNA sequences using the Illumina MiSeq platform. J Microbiol. 53, 60–69 (2015). https://doi.org/10.1007/s12275-015-4601-y

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  • DOI: https://doi.org/10.1007/s12275-015-4601-y

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