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Design Review 2012 32 Bit Torrent


Precise assessment of the composition of the human gut microbiota crucially depends on the reliability and specificity of the PCR primers employed to amplify the 16S rRNA gene of specific groups of the bacterial community. Comparative assays were performed by amplification reactions using the same samples as templates and either different sets of previously described PCR primers targeting bacterial [32] [22], [27], [28], [32], [33], or the newly designed PCR primer pair (Probio_Uni/Probio_Rev) aimed at amplification of the 16S rRNA gene of key gut microbiota members such as those belonging to the genera Bifidobacterium, Bacteroides, Collinsella, Prevotella, Escherichia, Blautia, Faecalibacterium, Klebsiella and Methanobrevibacter. Notably, no or very little PCR-mediated amplification product was obtained when DNA, extracted from commonly encountered human gut bifidobacterial species (Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium catenulatum/Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum and Bifidobacterium gallicum) (for review see [34]), was used as a template and employing Bact-8F/Bact-1510R, ENV1/ENV2 and Bact-8F/1319R primer combinations. The dissection by quantitative real-time qPCR of the microbial composition according to several common intestinal microbiota taxa (e.g., Bifidobacteriaceae, Enterobacteriaceae, Enterococcaceae, Lactobacillales, Bacteroidetes group, and Atopobium group) of amplicons obtained from fecal DNA extracted using the method involving an enzymatic lysis based on previously published PCR primers [22], [27], [28], [32], [33] yielded very different microbial profiles (Fig. 2). In particular, the microbial taxon Bifidobacterium appears to be completely absent when employing primer combinations ENV1/ENV2 and Bact-8F/Bact-1510R [22], [33]. This is consistent with these primers exhibiting the lowest level of homology with the corresponding 16S rRNA gene sequences of members of the taxa Bifidobacteriaceae, Lactobacillus and Enterobacteriaceae (Fig. 1). Altogether these results lead us to conclude that the apparent lack of bifidobacterial sequences from certain previously published 16S rRNA gene profiling gut microbiota studies is in part due to PCR primers that were biased against key members of the gut microbiota such as bifidobacteria. Thus, many of the published studies describing the bacterial composition of the infant gut seem to have significantly underestimated the incidence and diversity of these commensals.




Design Review 2012 32 Bit Torrent



Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.


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