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

다국어 초록 (Multilingual Abstract)

The cost of DNA sequencing has decreased due to advancementsin Next Generation Sequencing. The number of sequencesobtained from the Illumina platform is large, use ofthis platform can reduce costs more than the 454 pyrosequencer.
However, the Illumina platform has other challenges,including bioinformatics analysis of large numbersof sequences and the need to reduce erroneous nucleotidesgenerated at the 3􍿁-ends of the sequences. These erroneoussequences can lead to errors in analysis of microbial communities.
Therefore, correction of these erroneous sequencesis necessary for accurate taxonomic identification. Severalstudies that have used the Illumina platform to perform metagenomicanalyses proposed curating pipelines to increaseaccuracy. In this study, we evaluated the likelihood of obtainingan erroneous microbial composition using the MiSeq250 bp paired sequence platform and improved the pipelineto reduce erroneous identifications. We compared differentsequencing conditions by varying the percentage of controlphiX added, the concentration of the sequencing library, andthe 16S rRNA gene target region using a mock communitysample composed of known sequences. Our recommendedmethod corrected erroneous nucleotides and improved identificationaccuracy. Overall, 99.5% of the total reads shared95% similarity with the corresponding template sequencesand 93.6% of the total reads shared over 97% similarity. Thisindicated that the MiSeq platform can be used to analyze microbialcommunities at the genus level with high accuracy.
The improved analysis method recommended in this studycan be applied to amplicon studies in various environmentsusing high-throughput reads generated on the MiSeq platform.

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