Frank DN.. BARCRAWL and BARTAB: software tools for the design and implementation of barcoded primers for highly multiplexed DNA sequencing. BMC Bioinformatics 10: 362

Department of Molecular, University of Colorado, Boulder, CO 80309, USA.
BMC Bioinformatics (Impact Factor: 2.58). 10/2009; 10(1):362. DOI: 10.1186/1471-2105-10-362
Source: PubMed


Advances in automated DNA sequencing technology have greatly increased the scale of genomic and metagenomic studies. An increasingly popular means of increasing project throughput is by multiplexing samples during the sequencing phase. This can be achieved by covalently linking short, unique "barcode" DNA segments to genomic DNA samples, for instance through incorporation of barcode sequences in PCR primers. Although several strategies have been described to insure that barcode sequences are unique and robust to sequencing errors, these have not been integrated into the overall primer design process, thus potentially introducing bias into PCR amplification and/or sequencing steps.
Barcrawl is a software program that facilitates the design of barcoded primers, for multiplexed high-throughput sequencing. The program bartab can be used to deconvolute DNA sequence datasets produced by the use of multiple barcoded primers. This paper describes the functions implemented by barcrawl and bartab and presents a proof-of-concept case study of both programs in which barcoded rRNA primers were designed and validated by high-throughput sequencing.
Barcrawl and bartab can benefit researchers who are engaged in metagenomic projects that employ multiplexed specimen processing. The source code is released under the GNU general public license and can be accessed at

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    • "Sequence reads were assigned to sample of origin using the bar code sequence added during PCR and screened for basic quality defects (short sequences <200 nucleotides [nt] in length; >1 nt ambiguity, best read with quality ≥20 over a 10 nt moving window ) by the software program BARTAB [11]. Potential chimeras identified with Uchime (usearch6.0.203 i86linux32) [8] using the Schloss Silva reference sequences [31] were removed from subsequent analysis. "
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    ABSTRACT: Opportunistic pathogens, including Legionella spp. and non-tuberculous mycobacteria, can thrive in building hot water systems despite municipal and traditional on-site chlorine disinfection. Monochloramine is a relatively new approach to on-site disinfection, but the microbiological impact of on-site chloramine use has not been well studied. We hypothesized that comparison of the microbial ecology associated with monochloramine treatment versus no on-site treatment would yield highly dissimilar bacterial communities. Hot water samples were collected monthly from 7 locations for three months from two buildings in a Pennsylvania hospital complex supplied with common municipal water: (1) a hospital administrative building (no on-site treatment) and (2) an adjacent acute-care hospital treated on-site with monochloramine to control Legionella spp. Water samples were subjected to DNA extraction, rRNA PCR, and 454 pyrosequencing. Stark differences in the microbiome of the chloraminated water and the control were observed. Bacteria in the treated samples were primarily Sphingomonadales and Limnohabitans, whereas Flexibacter and Planctomycetaceae predominated in untreated control samples. Serendipitously, one sampling month coincided with dysfunction of the on-site disinfection system that resulted in a Legionella bloom detected by sequencing and culture. This study also demonstrates the potential utility of high-throughput DNA sequencing to monitor microbial ecology in water systems. Copyright © 2015 Elsevier GmbH. All rights reserved.
    Systematic and Applied Microbiology 03/2015; 38(3). DOI:10.1016/j.syapm.2015.02.006 · 3.28 Impact Factor
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    • "8 bp MIDs were designed using the barcrawl program (Frank 2009), which provided 755 potential MIDs with the following characteristics and provided in the supplementary material S1: at least three differences between all MIDs, no homopolymers of more than two nucleotides, no hairpins or heteroduplexes of more than four nucleotides, no possible convergence of MIDs to identical sequences through 1 bp deletion. For the test experiment described here, a combination of 100 MIDs (listed in supplementary material S2) was selected to ensure an approximately balanced base composition at each position (even ratios of A, C, G and T). "
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    ABSTRACT: Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.
    Genetica 02/2015; 143(2). DOI:10.1007/s10709-015-9828-3 · 1.40 Impact Factor
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    • "The hypervariable V3-V5 region of the 16S rRNA gene was amplified using the primer set 341F (5 CCTACGGGAGGCAGCAG 3 ) and 907R (5 CCGTCAATTCCTTTRAGTTT 3 ). A unique 8- nucleotide barcode was added to both primers for multiplexed pyrosequencing using barcrawl (Pourmand et al., 2002; Frank, 2009). Each 20-µL PCR reaction consisted of 4 µL of 5 × Phusion "
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    ABSTRACT: Marine sponges play important roles in benthic environments and are sensitive to environmental stresses. Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants since the 1970s and are cytotoxic and genotoxic to organisms. In the present study, we studied the short-period effect of PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) treatment on the community structure and functional gene composition of the bacterial community inhabiting the marine sponge Haliclona cymaeformis. Our results showed that the bacterial community shifted from an autotrophic bacteria-dominated community to a heterotrophic bacteria-dominated community in response to PBDE-47 in a time- and concentration-dependent manner. A potentially symbiotic sulfur-oxidizing bacterium (SOB) was dominant (>80% in abundance) in the untreated sponge. However, exposure to a high concentration (1 µg/L) of PBDE-47 caused a substantial decrease in the potential symbiont and an enrichment of heterotrophic bacteria like Clostridium. A metagenomic analysis showed a selective effect of the high concentration treatment on the functional gene composition of the enriched heterotrophic bacteria, revealing an enrichment for the functions responsible for DNA repair, multidrug efflux pumping, and bacterial chemotaxis and motility. This study demonstrated that PBDE-47 induced a shift in the composition of the community and functional genes in the sponge-associated bacterial community, revealing the selective effect of PBDE-47 treatment on the functions of the bacterial community in the microenvironment of the sponge.
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