bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/010058

Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the Illumina platform

Lucas Sinclair·Omneya Ahmed Osman·Stefan Bertilsson·Alexander Eiler
View original source ↗Export metadata ↓

Abstract

As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity.\n\nUsing different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner - with the exception for evenness estimates where correspondence between the methods was low.\n\nWe further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods.\n\nOur procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.

Source connections

Explore related subjects

Molecular BiologyPreprint

Keep this discovery

BibTeXRIS

Lucas Sinclair, Omneya Ahmed Osman, Stefan Bertilsson, Alexander Eiler. 2014-10-06. Microbial community composition and diversity via 16S rRNA gene amplicons: evaluating the Illumina platform. https://doi.org/10.1101/010058

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2016

Lysine as a potential low molecular weight angiogen: its clinical, experimental and in-silico validation- A brief study

Globally, the area of angiogenesis is dominated by investigations on anti-angiogenic agents and processes, due to its role in metastatic cancer treatment. Although, the area of ischemic tissue reperfusion is having much bigger demand and foot-mark. Following clinical failure of VEGF (Vascular endothelial growth factor) as a potential agent for induction of a controlled angiogenic response in ischemic tissues and organs, the progress is reasonably quiet as for new low molecular weight (LMW) angiogen molecules and their clinical applications are concerned. Basic amino acid Lysine has been observed to have profound angiogenic property in ischemic tissues, which is controlled, reproducible, time bound and without any accompanying reperfusion damage. In this study, the basic amino acid Lysine has been suggested as a LMW-angiogen, where it has been proposed to have a molecular binding property between VEGF and VEGF receptor (VEGFR). Here, the molecular adhesive hypothesis is being probed and confirmed both in the clinical and lab conditions through induced angiogenic response in tissue repair and in chick chorio allantoic membrane (CAM), respectively; and in dry-docking experiments (in-silico studies).

Molecular Biology↗
bioRxiv2016

Evolving Notch polyQ tracts reveal possible solenoid interference elements

Polyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved \"polyQ insertion slots\". Notch polyQ insertion slots are: (i) identifiable by an amphipathic \"slot leader\" motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and {beta}-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results strongly suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment are coupled proteodynamic modulators of solenoids.\n\nSIGNIFICANCENeurodegenerative disorders are often caused by expanded polyglutamine (polyQ) tracts embedded in the disordered regions of regulatory proteins, which are difficult to characterize structurally. To identify functional principles underlying polyQ tracts in disordered regulatory domains, I analyze evolution of the Notch protein, which can generate polyQ-related alleles causing neurodevelopmental defects. I show that Notch evolves polyQ tracts that come and go in a few conserved \"polyQ insertion slots\". Several features suggest these slots are ankyrin repeat (AR) interference elements, which dynamically compete with the delicate solenoid formed by Notch. Huntingtin, whose polyQ expansions causes Huntingtons Disease in humans, also has solenoid-forming modules and polyQ insertion slots, suggesting a common architectural principle underlies solenoid-forming polyQ-rich proteins.

Molecular Biology↗
bioRxiv2016

Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC=\"FIGDIR/small/049122_fig1.gif\" ALT=\"Figure 1\">\nView larger version (35K):\norg.highwire.dtl.DTLVardef@1f0c15corg.highwire.dtl.DTLVardef@126a6fdorg.highwire.dtl.DTLVardef@9d6a2eorg.highwire.dtl.DTLVardef@a61f75_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1C_FLOATNO Cpf1 mediates processing of pre-crRNA. (a) Schematic of pre-crRNA processing for Cas9 and Cpf1. Cleavage sites indicated with red triangle. (b) In vitro processing of FnCpf1 pre-crRNA transcript (80 nM) with purified AsCpf1 or LbCpf1 protein ([~]320 nM). In the presence of Cpf1 nuclease the pre-crRNA was cleaved in a distinct pattern, indicating cleavage at similar sequence motifs. RNA molecules without Cpf1 DR features where not cleaved by Cpf1 (control RNA). (c) RNAseq analysis of FnCpf1 pre-crRNA cleavage products, as shown in (b). A high fraction of sequence reads smaller than 65nt are cleavage products of spacers flanked by DR sequences.\n\nC_FIG

Molecular Biology↗