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Genomic Sequencing of Single Microbial Cells from Environmental Samples

Description: Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.
Date: February 1, 2008
Creator: Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark & Lasken, Roger S.
Partner: UNT Libraries Government Documents Department

Analysis of Illumina Microbial Assemblies

Description: Since the emerging of second generation sequencing technologies, the evaluation of different sequencing approaches and their assembly strategies for different types of genomes has become an important undertaken. Next generation sequencing technologies dramatically increase sequence throughput while decreasing cost, making them an attractive tool for whole genome shotgun sequencing. To compare different approaches for de-novo whole genome assembly, appropriate tools and a solid understanding of both quantity and quality of the underlying sequence data are crucial. Here, we performed an in-depth analysis of short-read Illumina sequence assembly strategies for bacterial and archaeal genomes. Different types of Illumina libraries as well as different trim parameters and assemblers were evaluated. Results of the comparative analysis and sequencing platforms will be presented. The goal of this analysis is to develop a cost-effective approach for the increased throughput of the generation of high quality microbial genomes.
Date: May 28, 2010
Creator: Clum, Alicia; Foster, Brian; Froula, Jeff; LaButti, Kurt; Sczyrba, Alex; Lapidus, Alla et al.
Partner: UNT Libraries Government Documents Department

Final progress report, Construction of a genome-wide highly characterized clone resource for genome sequencing

Description: At TIGR, the human Bacterial Artificial Chromosome (BAC) end sequencing and trimming were with an overall sequencing success rate of 65%. CalTech human BAC libraries A, B, C and D as well as Roswell Park Cancer Institute's library RPCI-11 were used. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length {approx}460 bp for a total of 141 Mb covering {approx}4.7% of the genome. Over sixty percent of the clones have BAC end sequences (BESs) from both ends representing over five-fold coverage of the genome by the paired-end clones. The average phred Q20 length is {approx}400 bp. This high accuracy makes our BESs match the human finished sequences with an average identity of 99% and a match length of 450 bp, and a frequency of one match per 12.8 kb contig sequence. Our sample tracking has ensured a clone tracking accuracy of >90%, which gives researchers a high confidence in (1) retrieving the right clone from the BA C libraries based on the sequence matches; and (2) building a minimum tiling path of sequence-ready clones across the genome and genome assembly scaffolds.
Date: February 14, 2000
Creator: Nierman, William C.
Partner: UNT Libraries Government Documents Department

[DNA sample manipulation and automation.] Final report

Description: Goals for the project were the following: (1) Development of 1536 well based DNA sequencing platform, (2) Integration of the above device into the CuraGen MicroNiagara system, PerSeptive BioSystems Voyager MALDI Mass Spec and validation of results from the ABI 377 DNA sequencer, (3) Collaboration with the DOE Joint Genome Institute (JGI) to build Sequatron-like systems for DNA purification and DNA sequencing, and working with JGI to implement 1536 well system if required, and (4) Demonstration of the use of robotic systems through genomic sequencing of up to 1Mb DNA. Emphasis over the past year was on the following: DNA purification methods, module development (1536 well thermal cycling; Mass Spectrometry, 96 lane development & Automated gel loading; 96 lane ABI system; MALDI TOF mass spectrometry; automated gel loading system for the ABI), and helping the JGI with Sequatron-like systems.
Date: September 13, 2001
Creator: Hawkins, Trevor
Partner: UNT Libraries Government Documents Department

Contribution to Sequencing of the Deinococcus radiodurans Genome

Description: The stated goal of this project was to supply The Institute for Genomic Research (TIGR) with pure DNA from the bacterium Deinocmus radiodurans RI for purposes of complete genomic sequencing by TIGR. We subsequently decided to expand this project to include a second goal; this second goal was the development of a NotI chromosomal map of D. radiodurans R1 using Pulsed Field Gel Electrophoresis (PFGE).
Date: March 11, 1999
Creator: Minton, K.W.
Partner: UNT Libraries Government Documents Department

Advances in Genome Biology & Technology

Description: This year's meeting focused on the latest advances in new DNA sequencing technologies and the applications of genomics to disease areas in biology and biomedicine. Daytime plenary sessions highlighted cutting-edge research in areas such as complex genetic diseases, comparative genomics, medical sequencing, massively parallel DNA sequencing, and synthetic biology. Technical approaches being developed and utilized in contemporary genomics research were presented during evening concurrent sessions. Also, as in previous years, poster sessions bridged the morning and afternoon plenary sessions. In addition, for the third year in a row, the Advances in Genome Biology and Technology (AGBT) meeting was preceded by a pre-meeting workshop that aimed to provide an introductory overview for trainees and other meeting attendees. This year, speakers at the workshop focused on next-generation sequencing technologies, including their experiences, findings, and helpful advise for others contemplating using these platforms in their research. Speakers from genome centers and core sequencing facilities were featured and the workshop ended with a roundtable discussion, during which speakers fielded questions from the audience.
Date: December 1, 2007
Creator: Thomas J. Albert, Jon R. Armstrong, Raymond K. Auerback, W. Brad Barbazuk, et al.
Partner: UNT Libraries Government Documents Department

Sequencing and analysis of 10967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis

Description: Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection initiative. Here we present an analysis of 10967 clones (8049 from X. laevis and 2918 from X. tropicalis). The clone set contains 2013 orthologs between X. laevis and X. tropicalis as well as 1795 paralog pairs within X. laevis. 1199 are in-paralogs, believed to have resulted from an allotetraploidization event approximately 30 million years ago, and the remaining 546 are likely out-paralogs that have resulted from more ancient gene duplications, prior to the divergence between the two species. We do not detect any evidence for positive selection by the Yang and Nielsen maximum likelihood method of approximating d{sub N}/d{sub S}. However, d{sub N}/d{sub S} for X. laevis in-paralogs is elevated relative to X. tropicalis orthologs. This difference is highly significant, and indicates an overall relaxation of selective pressures on duplicated gene pairs. Within both groups of paralogs, we found evidence of subfunctionalization, manifested as differential expression of paralogous genes among tissues, as measured by EST information from public resources. We have observed, as expected, a higher instance of subfunctionalization in out-paralogs relative to in-paralogs.
Date: October 31, 2005
Creator: Morin, R D; Chang, E; Petrescu, A; Liao, N; Kirkpatrick, R; Griffith, M et al.
Partner: UNT Libraries Government Documents Department

Development of High Throughput Process for Constructing 454 Titanium and Illumina Libraries

Description: We have developed two processes with the Biomek FX robot to construct 454 titanium and Illumina libraries in order to meet the increasing library demands. All modifications in the library construction steps were made to enable the adaptation of the entire processes to work with the 96-well plate format. The key modifications include the shearing of DNA with Covaris E210 and the enzymatic reaction cleaning and fragment size selection with SPRI beads and magnetic plate holders. The construction of 96 Titanium libraries takes about 8 hours from sheared DNA to ssDNA recovery. The processing of 96 Illumina libraries takes less time than that of the Titanium library process. Although both processes still require manual transfer of plates from robot to other work stations such as thermocyclers, these robotic processes represent about 12- to 24-folds increase of library capacity comparing to the manual processes. To enable the sequencing of many libraries in parallel, we have also developed sets of molecular barcodes for both library types. The requirements for the 454 library barcodes include 10 bases, 40-60percent GC, no consecutive same base, and no less than 3 bases difference between barcodes. We have used 96 of the resulted 270 barcodes to construct libraries and pool to test the ability of accurately assigning reads to the right samples. When allowing 1 base error occurred in the 10 base barcodes, we could assign 99.6percent of the total reads and 100percent of them were uniquely assigned. As for the Illumina barcodes, the requirements include 4 bases, balanced GC, and at least 2 bases difference between barcodes. We have begun to assess the ability to assign reads after pooling different number of libraries. We will discuss the progress and the challenges of these scale-up processes.
Date: May 28, 2010
Creator: Deshpande, Shweta; Hack, Christopher; Tang, Eric; Malfatti, Stephanie; Ewing, Aren; Lucas, Susan et al.
Partner: UNT Libraries Government Documents Department

Illumina Production Sequencing at the DOE Joint Genome Institute - Workflow and Optimizations

Description: The U.S. Department of Energy (DOE) Joint Genome Institute?s (JGI) Production Sequencing group is committed to the generation of high-quality genomic DNA sequence to support the DOE mission areas of renewable energy generation, global carbon management, and environmental characterization and clean-up. Within the JGI?s Production Sequencing group, the Illumina Genome Analyzer pipeline has been established as one of three sequencing platforms, along with Roche/454 and ABI/Sanger. Optimization of the Illumina pipeline has been ongoing with the aim of continual process improvement of the laboratory workflow. These process improvement projects are being led by the JGI?s Process Optimization, Sequencing Technologies, Instrumentation& Engineering, and the New Technology Production groups. Primary focus has been on improving the procedural ergonomics and the technicians? operating environment, reducing manually intensive technician operations with different tools, reducing associated production costs, and improving the overall process and generated sequence quality. The U.S. DOE JGI was established in 1997 in Walnut Creek, CA, to unite the expertise and resources of five national laboratories? Lawrence Berkeley, Lawrence Livermore, Los Alamos, Oak Ridge, and Pacific Northwest ? along with HudsonAlpha Institute for Biotechnology. JGI is operated by the University of California for the U.S. DOE.
Date: June 18, 2010
Creator: Tarver, Angela; Fern, Alison; Diego, Matthew San; Kennedy, Megan; Zane, Matthew; Daum, Christopher et al.
Partner: UNT Libraries Government Documents Department

Molluscan Evolutionary Genomics

Description: In the last 20 years there have been dramatic advances in techniques of high-throughput DNA sequencing, most recently accelerated by the Human Genome Project, a program that has determined the three billion base pair code on which we are based. Now this tremendous capability is being directed at other genome targets that are being sampled across the broad range of life. This opens up opportunities as never before for evolutionary and organismal biologists to address questions of both processes and patterns of organismal change. We stand at the dawn of a new 'modern synthesis' period, paralleling that of the early 20th century when the fledgling field of genetics first identified the underlying basis for Darwin's theory. We must now unite the efforts of systematists, paleontologists, mathematicians, computer programmers, molecular biologists, developmental biologists, and others in the pursuit of discovering what genomics can teach us about the diversity of life. Genome-level sampling for mollusks to date has mostly been limited to mitochondrial genomes and it is likely that these will continue to provide the best targets for broad phylogenetic sampling in the near future. However, we are just beginning to see an inroad into complete nuclear genome sequencing, with several mollusks and other eutrochozoans having been selected for work about to begin. Here, we provide an overview of the state of molluscan mitochondrial genomics, highlight a few of the discoveries from this research, outline the promise of broadening this dataset, describe upcoming projects to sequence whole mollusk nuclear genomes, and challenge the community to prepare for making the best use of these data.
Date: December 1, 2005
Creator: Simison, W. Brian & Boore, Jeffrey L.
Partner: UNT Libraries Government Documents Department

Large Gap Size Paired-end Library Construction for Second Generation Sequencing

Description: Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 8 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 PE Library protocol. With the increasing fragment sizes, we found a general trend of decreasing library quality in several areas. First, redundant reads and reads containing multiple loxP linkers increase when the average fragment size increases. Second, the contamination of short distance pairs (<10Kb) increases as the fragment size increases. Third, chimeric rate increases with the increasing fragment sizes. We have modified several steps to improve the quality of the long span PE libraries. The modification includes (1) the use of special PFGE program to reduce small fragment contamination; (2) the increase of DNA samples in the circularization step and prior to the PCR to reduce redundant reads; and (3) the decrease of fragment size in the double SPRI size selection to get a higher frequency of LoxP linker containing reads. With these modifications we have generated large gap size PE libraries with a much better quality.
Date: May 28, 2010
Creator: Peng, Ze; Hamilton, Matthew; Froula, Jeff; Ewing, Aren; Foster, Brian & Cheng, Jan-Fang
Partner: UNT Libraries Government Documents Department

The"minimum information about an environmental sequence" (MIENS) specification

Description: We present the Genomic Standards Consortium's (GSC) 'Minimum Information about an ENvironmental Sequence' (MIENS) standard for describing marker genes. Adoption of MIENS will enhance our ability to analyze natural genetic diversity across the Tree of Life as it is currently being documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.
Date: October 15, 2010
Creator: Yilmaz, P.; Kottmann, R.; Field, D.; Knight, R.; Cole, J.R.; Amaral-Zettler, L. et al.
Partner: UNT Libraries Government Documents Department

Progress towards DNA sequencing at the single molecule level

Description: We describe progress towards sequencing DNA at the single molecule level. Our technique involves incorporation of fluorescently tagged nucleotides into a targeted sequence, anchoring the labeled DNA strand in a flowing stream, sequential exonuclease digestion of the DNA strand, and efficient detection and identification of single tagged nucleotides. Experiments demonstrating strand specific exonuclease digestion of fluorescently labeled DNA anchored in flow as well as the detection of single cleaved fluorescently tagged nucleotides from a small number of anchored DNA fragments axe described. We find that the turnover rate of Esherichia coli exonuclease III on fluorescently labeled DNA in flow at 36{degree}C is {approximately}7 nucleotides per DNA strand per second, which is approximately the same as that measured for this enzyme on native DNA under static, saturated (excess enzyme) conditions. Experiments demonstrating the efficient detection of single fluorescent molecules delivered electrokinetically to a {approximately}3 pL probe volume are also described.
Date: December 1, 1995
Creator: Goodwin, P.M.; Affleck, R.L. & Ambrose, W.P.
Partner: UNT Libraries Government Documents Department

Gene prediction by pattern recognition and homology search

Description: This paper presents an algorithm for combining pattern recognition-based exon prediction and database homology search in gene model construction. The goal is to use homologous genes or partial genes existing in the database as reference models while constructing (multiple) gene models from exon candidates predicted by pattern recognition methods. A unified framework for gene modeling is used for genes ranging from situations with strong homology to no homology in the database. To maximally use the homology information available, the algorithm applies homology on three levels: (1) exon candidate evaluation, (2) gene-segment construction with a reference model, and (3) (complete) gene modeling. Preliminary testing has been done on the algorithm. Test results show that (a) perfect gene modeling can be expected when the initial exon predictions are reasonably good and a strong homology exists in the database; (b) homology (not necessarily strong) in general helps improve the accuracy of gene modeling; (c) multiple gene modeling becomes feasible when homology exists in the database for the involved genes.
Date: May 1, 1996
Creator: Xu, Y. & Uberbacher, E.C.
Partner: UNT Libraries Government Documents Department

DNA sequence pattern recognition methods in GRAIL

Description: The goal of the GRAIL project has been to create a comprehensive analysis environment where a host of questions about genes and genome structure can be answered as quickly and accurately as possible. Constructing this system has entailed solving a number of significant technical challenges including: (a) making coding recognition in sequence more sensitive and accurate, (b) compensating for isochore base compositional effects in coding prediction, (c) developing methods to determine which parts of each strand of a long genomic DNA are the coding strand, (d) improving the accuracy of splice site prediction and recognizing non-consensus sites, and (e) recognizing variable regulatory structures such as polymerase II promoters. An additional challenge has been to construct algorithms which compensate for the deleterious effects of insertion or deletion (indel) errors in the coding region recognition process. This paper addresses progress on these technical issues and the current state of sequence feature recognition methods.
Date: December 31, 1995
Creator: Uberbacher, E.C.; Xu, Ying; Shah, M.; Matis, S.; Guan, X. & Mural, R.J.
Partner: UNT Libraries Government Documents Department

The Functional Genomics Initiative at Oak Ridge National Laboratory

Description: The Functional Genomics Initiative at the Oak Ridge National Laboratory integrates outstanding capabilities in mouse genetics, bioinformatics, and instrumentation. The 50 year investment by the DOE in mouse genetics/mutagenesis has created a one-of-a-kind resource for generating mutations and understanding their biological consequences. It is generally accepted that, through the mouse as a surrogate for human biology, we will come to understand the function of human genes. In addition to this world class program in mammalian genetics, ORNL has also been a world leader in developing bioinformatics tools for the analysis, management and visualization of genomic data. Combining this expertise with new instrumentation technologies will provide a unique capability to understand the consequences of mutations in the mouse at both the organism and molecular levels. The goal of the Functional Genomics Initiative is to develop the technology and methodology necessary to understand gene function on a genomic scale and apply these technologies to megabase regions of the human genome. The effort is scoped so as to create an effective and powerful resource for functional genomics. ORNL is partnering with the Joint Genome Institute and other large scale sequencing centers to sequence several multimegabase regions of both human and mouse genomic DNA, to identify all the genes in these regions, and to conduct fundamental surveys to examine gene function at the molecular and organism level. The Initiative is designed to be a pilot for larger scale deployment in the post-genome era. Technologies will be applied to the examination of gene expression and regulation, metabolism, gene networks, physiology and development.
Date: December 31, 1997
Creator: Johnson, Dabney; Justice, Monica; Beattle, Ken; Buchanan, Michelle; Ramsey, Michael; Ramsey, Rose et al.
Partner: UNT Libraries Government Documents Department

A fast look-up algorithm for detecting repetitive DNA sequences

Description: We have presented a fast linear time algorithm for recognizing tandem repeats. Our algorithm is a one pass algorithm. No information about the periodicity of tandem repeats is needed. The use of the indices calculated from non-continuous and overlapping {kappa}-tuples allow tandem repeats with insertions and deletions to be recognized.
Date: December 31, 1996
Creator: Guan, X. & Uberbacher, E.C.
Partner: UNT Libraries Government Documents Department