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

1 Follower
40 Reads
  • Source
    • "The PCR reaction mixture contained unique primer pair combinations for each sample, each with a different barcode sequence at their 5 0 end (Parameswaran et al. 2007). Six base pair long barcode sequences for forward and reverse primers were created with the Barcrawl program (Frank 2009). PCR was performed with the Phusion Hot Start High Fidelity Polymerase (Thermo Fisher Scientific, Waltham , MA, USA) reaction mixture according to the manufacturer's instructions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This two-week anaerobic batch study evaluated 2,4,6-trinitrotoluene (TNT) removal efficiency from industrial pink water by (1) adsorption on low-cost adsorbent pine bark, and (2) adsorption coupled with TNT biotransformation by specialised microbial communities. Samples of the supernatant and acetonitrile extracts of pine bark were analysed by HPLC, while the composition of the bacterial community of the experimental batches, inocula and pine bark were profiled by high-throughput sequencing the V6 region of the bacterial 16S rRNA gene. Integrated adsorption and biotransformation proved to be the most efficient method for TNT removal from pink water. The type of applied inoculum had a profound effect on TNT removal efficiencies and microbial community structures, which were dominated by phylotypes belonging to the Enterobacteriaceae family. The analysis of acetonitrile extracts of pine bark supported the hypothesis that the microbial community indigenous to pine bark has the ability to degrade TNT.
    Biodegradation 07/2015; 26(5):1-12. DOI:10.1007/s10532-015-9740-7 · 2.34 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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
Show more

Preview (2 Sources)

40 Reads
Available from