![]() ![]() A few studies used PGM for medical diagnosis with marker genes ( 15, – 17). To date, sequencing platform comparisons or case studies employing the Ion Torrent PGM were focused mainly on microbial whole-genome sequencing ( 6, 7, 10, 11) or on bacterial community structure analysis using 16S rRNA gene multiplexing tags ( 12, – 14). This information can then be used to determine the proper parameters and filters for parsing raw sequence data to minimize the impact of these errors on biological conclusions ( 8, 9). The higher error rate has been problematic for microbial ecology, but by parallel sequencing of mock communities of known composition, the type and extent of insertion, deletion, or substitution errors can be characterized. Sequencing quality (error type and rate) and length have suffered with NGS platforms ( 3, 6, 7) compared to Sanger sequencing, but the greatly improved sequence yields and reduced cost have resulted in NGS dominating use. Next-generation sequencing (NGS) technologies have evolved rapidly in the years since pyrosequencing was initially launched in 2005 ( 1), followed by light-imaging Illumina ( 2, 3) HiSeq and MiSeq and by the post-light-sequencing Ion Torrent ( 4, 5) Personal Genome Machine (PGM) and Proton. The current crop type, as well as the crop rotation history, significantly influenced the composition of the soil diazotrophic community detected. For the cropping system analyses, a total of 6,182 unique NifH operational taxonomic units at 5% amino acid dissimilarity were obtained. Significant differences among platforms were identified, although the diversity indices and overall site-based conclusions remained similar. The PGM produced higher frameshift rates than pyrosequencing that were corrected by the RDP FrameBot tool. Compared with the 400-bp kit, the Hi-Q kit reduced indel rates by 28 to 59% and produced one to seven times more reads acceptable for downstream analyses. Using our suggested filters from mock community analyses, the overall error rates were 0.62, 0.36, and 0.39% per base for chips 318 and 314 with the 400-bp kit and chip 318 with the Hi-Q chemistry, respectively. The PGM was then used to assess differences in nifH-harboring bacterial community structure among four corn-based cropping systems. To address this, we compared sequence profiles across available PGM chips and chemistries and with 454 pyrosequencing data by determining error types and rates and diazotrophic community structures. ![]() The quality of PGM sequences has not been reassessed, and results have not been compared in the context of a gene-targeted microbial ecology study. ![]() The sequencing chips and kits of the Ion Torrent Personal Genome Machine (PGM), which employs semiconductor technology to measure pH changes in polymerization events, have recently been upgraded. ![]()
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