DOCSLIB.ORG

Bioinformatics for Whole-Genome Shotgun Sequencing of Microbial Communities

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bioinformatics for Whole-Genome Shotgun Sequencing of Microbial Communities Review Bioinformatics for Whole-Genome Shotgun Sequencing of Microbial Communities Kevin Chen*, Lior Pachter* ABSTRACT sequencing of the entire clone library has emerged as a third approach to metagenomics. Unlike previous approaches, he application of whole-genome shotgun sequencing which typically study a single gene or individual genomes, this to microbial communities represents a major approach offers a more global view of the community, T development in metagenomics, the study of allowing us to better assess levels of phylogenetic diversity uncultured microbes via the tools of modern genomic and intraspecies polymorphism, study the full gene analysis. In the past year, whole-genome shotgun sequencing complement and metabolic pathways in the community, and projects of prokaryotic communities from an acid mine in some cases, reconstruct near-complete genome sequences. biofilm, the Sargasso Sea, Minnesota farm soil, three deep-sea WGS also has the potential to discover new genes that are too whale falls, and deep-sea sediments have been reported, diverged from currently known genes to be amplified with adding to previously published work on viral communities PCR, or heterologously expressed in common hosts, and is from marine and fecal samples. The interpretation of this especially important in the case of viral communities because new kind of data poses a wide variety of exciting and difficult of the lack of a universal gene analogous to 16S. bioinformatics problems. The aim of this review is to Nine shotgun sequencing projects of various communities introduce the bioinformatics community to this emerging have been completed to date (Table 1). The biological insights field by surveying existing techniques and promising new from these studies have been well-reviewed elsewhere [3,6]. approaches for several of the most interesting of these Here, we highlight just two studies that exemplify the exciting computational problems. Introduction Table 1. Published Microbial Community Shotgun Sequencing Projects Metagenomics is the application of modern genomics techniques to the study of communities of microbial Type Community Species Sequence (Mbp) Reference organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species Prokaryote Acid mine biofilm 5 75 18 [1–6]. The field has its roots in the culture-independent Sargasso Sea 1,800 1,600 19 retrieval of 16S rRNA genes, pioneered by Pace and Minnesota soil 3,000 100 21 colleagues two decades ago [7]. Since then, metagenomics has Whale falls 150 25 21 Deep-sea sedimenta ? 111 22 revolutionized microbiology by shifting focus away from Viralb Sea water 374-7114 0.74 30 clonal isolates towards the estimated 99% of microbial Marine sediment 103–106 0.7 71 species that cannot currently be cultivated [8,9]. Human feces 1,200 0.037 54 A typical metagenomics project begins with the Equine feces 233 0.018 72 construction of a clone library from DNA sequence retrieved from an environmental sample. Clones are then selected for aThe deep-sea sediment project used an additional 20 Mbp of fosmid sequence and also a filter to reduce the complexity of the community prior to sequencing. sequencing using either functional or sequence-based bThe viral projects used linker-amplified shotgun libraries. screens. In the functional approach, genes retrieved from the DOI: 10.1371/journal.pcbi.0010024.t001 environment are heterologously expressed in a host, such as Escherichia coli, and sophisticated functional screens employed to detect clones expressing functions of interest [10–12]. This Citation: Chen K, Pachter L (2005) Bioinformatics for whole-genome shotgun approach has produced many exciting discoveries and sequencing of microbial communities. PLoS Comp Biol 1(2): e24. spawned several companies aiming to retrieve marketable Copyright: Ó 2005 Chen and Pachter. This is an open-access article distributed natural products from the environment (e.g., Diversa [http:// under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the www.diversa.com] and Cubist Pharmaceuticals [http:// original author and source are credited. www.cubist.com]). In the sequence-based approach, clones Abbreviations: HMM, hidden Markov model; MSA, multiple sequence alignment; are selected for sequencing based on the presence of either WGS, whole-genome shotgun phylogenetically informative genes, such as 16S, or other Kevin Chen is in the Department of Electrical Engineering and Computer Science genes of biological interest [13–17]. The most prominent and Lior Pachter is in the Department of Mathematics at the University of discovery from this approach thus far is the discovery of the California, Berkeley, California, United States of America. proteorhodopsin gene from a marine community [14]. *To whom correspondence should be addressed. E-mail: [email protected] Recently, facilitated by the increasing capacity of (KC), [email protected] (LP) sequencing centers, whole-genome shotgun (WGS) DOI: 10.1371/journal.pcbi.0010024 PLoS Computational Biology | www.ploscompbiol.org0106 July 2005 | Volume 1 | Issue 2 | e24 possibilities of the approach. The acid mine biofilm from a different individual in the population. Second, highly community [18] is an extremely simple model system, conserved sequence shared between different species can consisting of only four dominant species, so a relatively seed contigs and cause false overlaps. In some communities, miniscule amount of shotgun sequencing (75 Mbp) was even phylogenetically distant genomes can share a large enough to produce two near-complete genome sequences number of genes [29]. Careful study of the optimal overlap and detailed information about metabolic pathways and parameters for separating out sequences at different strain-level polymorphism. At the other end of the spectrum, phylogenetic distances is important, and has been carried out the Sargasso Sea community is extremely complex, for viral communities [30], but not yet for prokaryotes. containing more than 1,800 species [19,20]. Nonetheless, with The assembly of communities has strong similarities to the an enormous amount of sequencing (1.6 Gbp), vast amounts assembly of highly polymorphic diploid eukaryotes, such as of previously unknown diversity were discovered, including Ciona savigny [26] and Candida albicans [31], if we view over 1.2 million new genes, 148 new species, and numerous prokaryotic strains as analogous to eukaryotic haplotypes. new rhodopsin genes. These results were especially surprising The main difference is that in a microbial community, the given how well the community had been studied previously, number of strains is unknown and potentially large, and their and suggest that equally large amounts of biological diversity relative abundance is also unknown and potentially skewed, await future discovery. while in most eukaryotes we know a priori the number of In this review, we survey several of the most interesting haplotypes and their relative abundance. This disadvantage is computational problems that arise from WGS sequencing of mitigated somewhat by the small size and relative lack of communities. Traditional approaches to classic repetitive sequence in prokaryotic and viral genomes, so that bioinformatics problems such as assembly, gene finding, and the issue of distinguishing alleles from paralogs and phylogeny need to be reconsidered in light of this new kind of polymorphism from repetitive sequence is less acute. data, while new problems need to be addressed, including Thus far, both community assembly and polymorphic how to compare communities, how to separate sequence eukaryotic assembly have been performed by running a from different organisms in silico, and how to model single-genome assembler, such as the Celera assembler [32] or population structures using WGS assembly statistics. We Jazz [33], and then manually post-processing the resulting discuss all these problems and their connections to other scaffolds to correct assembly errors. Contigs erroneously split areas of bioinformatics, such as the assembly of highly apart because of polymorphism are reconnected, and contigs polymorphic genomes, gene expression analysis, and based on false overlaps are broken apart. Not surprisingly, ad supertree methods for phylogenetic reconstruction. hoc heuristics must be employed to adapt programs Although we have chosen to focus on the shotgun optimized for single-genome assembly: the Celera assembler, sequencing approach, we stress that this is only one piece of for instance, treats high-depth contigs associated with the exciting field of metagenomics, and that the integration abundant species as repetitive sequence. of other techniques such as large-insert clone sequencing, A promising direction for both these problems is co- microarray analysis, and proteomics will be vital to achieve a assembly, in which two very closely related genomes (or even comprehensive view of microbial communities. two assemblies of the same genome) are assembled concurrently, using alignment information to complement Assembling Communities mate-pair information in ordering scaffolds and correcting assembly errors in a structured, automated way. Thus far, the The retrieval of nearly complete genomes from the only published work on this problem is that of Sundararajan environment without prior lab cultivation is one of the most et al. [26], and even then, only for
Load more

Recommended publications
  • A Clone-Array Pooled Shotgun Strategy for Sequencing Large Genomes
    Downloaded from genome.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Perspective A Clone-Array Pooled Shotgun Strategy for Sequencing Large Genomes Wei-Wen Cai,1,2 Rui Chen,1,2 Richard A. Gibbs,1,2,5 and Allan Bradley1,3,4 1Department of Molecular and Human Genetics, 2Human Genome Sequencing Center, and 3Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA A simplified strategy for sequencing large genomes is proposed. Clone-Array Pooled Shotgun Sequencing (CAPSS) is based on pooling rows and columns of arrayed genomic clones,for shotgun library construction. Random sequences are accumulated,and the data are processed by sequential comparison of rows and columns to assemble the sequence of clones at points of intersection. Compared with either a clone-by-clone approach or whole-genome shotgun sequencing,CAPSS requires relatively few library constructions and only minimal computational power for a complete genome assembly. The strategy is suitable for sequencing large genomes for which there are no sequence-ready maps,but for which relatively high resolution STS maps and highly redundant BAC libraries are available. It is immediately applicable to the sequencing of mouse,rat,zebrafish, and other important genomes,and can be managed in a cooperative fashion to take advantage of a distributed international DNA sequencing capacity. Advances in DNA sequencing technology in recent years have Drosophila genome, and the computational requirements to greatly increased the throughput and reduced the cost of ge- perform the necessary pairwise comparisons increase approxi- nome sequencing. Sequencing of a complex genome the size mately as a square of the size of the genome (see Appendix).
    [Show full text]
  • Probabilities and Statistics in Shotgun Sequencing Shotgun Sequencing
    Probabilities and Statistics in Shotgun Sequencing Shotgun Sequencing. Before any analysis of a DNA sequence can take place it is first necessary to determine the actual sequence itself, at least as accurately as is reasonably possible. Unfortunately, technical considerations make it impossible to sequence very long pieces of DNA all at once. Current sequencing technologies allow accurate reading of no more than 500 to 800bp of contiguous DNA sequence. This means that the sequence of an entire genome must be assembled from collections of comparatively short subsequences. This process is called DNA sequence “assembly ”. One approach of sequence assembly is to produce the sequence of a DNA segment (called as a “contig”, or perhaps a genome) from a large number of randomly chosen sequence reads (many overlapping small pieces, each on the order of 500-800 bases). One difficulty of this process is that the locations of the fragments within the genome and with respect to each other are not generally known. However, if enough fragments are sequenced so that there will be many overlaps between them, the fragments can be matched up and assembled. This method is called “ shotgun sequencing .” Shotgun sequencing approaches, including the whole-genome shotgun approach, are currently a central part of all genome-sequencing efforts. These methods require a high level of automation in sample preparation and analysis and are heavily reliant on the power of modern computers. There is an interplay between substrates to be sequenced (genomes and their representation in clone libraries), the analytical tools for generating a DNA sequence, the sequencing strategies, and the computational methods.
    [Show full text]
  • BIOGRAPHICAL SKETCH NAME: Berger
    BIOGRAPHICAL SKETCH NAME: Berger, Bonnie eRA COMMONS USER NAME (credential, e.g., agency login): BABERGER POSITION TITLE: Simons Professor of Mathematics and Professor of Electrical Engineering and Computer Science EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable. Add/delete rows as necessary.) EDUCATION/TRAINING DEGREE Completion (if Date FIELD OF STUDY INSTITUTION AND LOCATION applicable) MM/YYYY Brandeis University, Waltham, MA AB 06/1983 Computer Science Massachusetts Institute of Technology SM 01/1986 Computer Science Massachusetts Institute of Technology Ph.D. 06/1990 Computer Science Massachusetts Institute of Technology Postdoc 06/1992 Applied Mathematics A. Personal Statement Advances in modern biology revolve around automated data collection and sharing of the large resulting datasets. I am considered a pioneer in the area of bringing computer algorithms to the study of biological data, and a founder in this community that I have witnessed grow so profoundly over the last 26 years. I have made major contributions to many areas of computational biology and biomedicine, largely, though not exclusively through algorithmic innovations, as demonstrated by nearly twenty thousand citations to my scientific papers and widely-used software. In recognition of my success, I have just been elected to the National Academy of Sciences and in 2019 received the ISCB Senior Scientist Award, the pinnacle award in computational biology. My research group works on diverse challenges, including Computational Genomics, High-throughput Technology Analysis and Design, Biological Networks, Structural Bioinformatics, Population Genetics and Biomedical Privacy. I spearheaded research on analyzing large and complex biological data sets through topological and machine learning approaches; e.g.
    [Show full text]
  • Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing
    THE SWISS DNA COMPANY Application Note · Next Generation Sequencing Metagenomics Analysis of Microbiota by Next Generation Shotgun Sequencing Understand the genetic potential of your community samples Provides you with hypothesis-free taxonomic analysis Introduction Microbiome studies are often based on as the dependency on a single gene to microbiome analysis overcoming said the sequencing of specific marker genes analyze a whole community, the intro- limitations. Whole genomic DNA of as for instance the prokaryotic 16S rRNA duction of PCR bias and the restriction a sample is isolated, fragmented and gene. Such amplicon-based approaches to describe only the taxonomic compo- finally sequenced. This allows a detailed are well established and widely used. sition and diversity. Shotgun metage- analysis of the taxonomic and functional However, they have limitations such nomics is a cutting edge technique for composition of a microbial community. Microsynth Competences and Services Microsynth offers a full shotgun to your project requirements, thus pro- requirements to guarantee scientifically metagenomics service for taxonomic viding you with just the right amount of reliable results for your project. After and functional profiling of clinical, envi- data to answer your questions. quality processing the reads are aligned ronmental or engineered microbiomes. Bioinformatics: Taxonomic and func- against a protein reference database The service covers the entire process tional analysis of metagenomic data- (e.g. NCBI nr). Taxonomic and functional from experimental design, DNA isola- sets is challenging. Alignment, binning binning and annotation are performed. tion, tailored sequencing to detailed and annotation of the large amounts The analysis is not restricted to prokar- and customized bioinformatics analy- of sequencing reads require expertise yotes but also includes eukaryotes and sis.
    [Show full text]
  • Lior Pachter Genome Informatics 2013 Keynote
    Stories from the Supplement Lior Pachter! Department of Mathematics and Molecular & Cell Biology! UC Berkeley! ! November 1, 2013! Genome Informatics, CSHL The Cufflinks supplements • C. Trapnell, B.A. Williams, G. Pertea, A. Mortazavi, G. Kwan, M.J. van Baren, S.L. Salzberg, B.J. Wold and L. Pachter, Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation, Nature Biotechnology 28, (2010), p 511–515. Supplementary Material: 42 pages. • C. Trapnell, D.G. Hendrickson, M. Sauvageau, L. Goff, J.L. Rinn and L. Pachter, Differential analysis of gene regulation at transcript resolution with RNA-seq, Nature Biotechnology, 31 (2012), p 46–53. Supplementary Material: 70 pages. A supplementary arithmetic progression? • C. Trapnell, B.A. Williams, G. Pertea, A. Mortazavi, G. Kwan, M.J. van Baren, S.L. Salzberg, B.J. Wold and L. Pachter, Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation, Nature Biotechnology 28, (2010), p 511–515. Supplementary Material: 42 pages. • C. Trapnell, D.G. Hendrickson, M. Sauvageau, L. Goff, J.L. Rinn and L. Pachter, Differential analysis of gene regulation at transcript resolution with RNA- seq, Nature Biotechnology, 31 (2012), p 46–53. Supplementary Material: 70 pages. • A. Roberts and L. Pachter, Streaming algorithms for fragment assignment, Nature Methods 10 (2013), p 71—73. Supplementary Material: 98 pages? The nature methods manuscript checklist The nature methods manuscript checklist Emperor Joseph II: My dear young man, don't take it too hard. Your work is ingenious. It's quality work. And there are simply too many notes, that's all.
    [Show full text]
  • Modeling and Analysis of RNA-Seq Data: a Review from a Statistical Perspective
    Modeling and analysis of RNA-seq data: a review from a statistical perspective Wei Vivian Li 1 and Jingyi Jessica Li 1;2;∗ Abstract Background: Since the invention of next-generation RNA sequencing (RNA-seq) technolo- gies, they have become a powerful tool to study the presence and quantity of RNA molecules in biological samples and have revolutionized transcriptomic studies. The analysis of RNA-seq data at four different levels (samples, genes, transcripts, and exons) involve multiple statistical and computational questions, some of which remain challenging up to date. Results: We review RNA-seq analysis tools at the sample, gene, transcript, and exon levels from a statistical perspective. We also highlight the biological and statistical questions of most practical considerations. Conclusion: The development of statistical and computational methods for analyzing RNA- seq data has made significant advances in the past decade. However, methods developed to answer the same biological question often rely on diverse statical models and exhibit dif- ferent performance under different scenarios. This review discusses and compares multiple commonly used statistical models regarding their assumptions, in the hope of helping users select appropriate methods as needed, as well as assisting developers for future method development. 1 Introduction RNA sequencing (RNA-seq) uses the next generation sequencing (NGS) technologies to reveal arXiv:1804.06050v3 [q-bio.GN] 1 May 2018 the presence and quantity of RNA molecules in biological samples. Since its invention, RNA- seq has revolutionized transcriptome analysis in biological research. RNA-seq does not require any prior knowledge on RNA sequences, and its high-throughput manner allows for genome-wide profiling of transcriptome landscapes [1,2].
    [Show full text]
  • Randomness Versus Order
    MILESTONES DOI: 10.1038/nrg2245 M iles Tone 1 0 Randomness versus order Whereas randomness is avoided used — in their opinion mistakenly The H. influenzae genome, in most experimental techniques, — direct sequencing strategies to however, was a mere DNA fragment it is fundamental to sequencing finish compared with the 1,500-fold longer approaches. In the race to sequence the last 10% of the bacteriophage λ ~3 billion base-pair human genome. the human genome, research groups sequence. In 1991, Al Edwards and In 1996, Craig Venter and colleagues had to choose between the random Thomas Caskey proposed a method proposed that the whole-genome whole-genome shotgun sequencing to maximize efficiency by minimiz- shotgun approach could be used to approach or the more ordered map- ing gap formation and redundancy: sequence the human genome owing based sequencing approach. sequence both ends (but not the to two factors: its past successes When Frederick Sanger and middle) of a long clone, rather than in assembling genomes and the colleagues sequenced the 48-kb the entirety of a short clone. development of bacterial artificial bacteriophage λ genome in 1982, Although the shotgun approach chromosomes (BAC) libraries, which the community was still undecided was now accepted for sequencing allowed large fragments of DNA to as to whether directed or random short stretches of DNA, map-based be cloned. sequencing strategies were better. techniques were still considered A showdown ensued, with the With directed strategies, DNA necessary for large genomes. Like biotechnology firm Celera Genomics sequences were broken down into the directed strategies, map-based wielding whole-genome shotgun ordered and overlapping fragments sequencing subdivided the genome sequencing and the International to build a map of the genome, and into ordered 40-kb fragments, which Human Genome Sequencing these fragments were then cloned were then sequenced using the Consortium wielding map-based and sequenced.
    [Show full text]
  • Shotgun Metagenomics, from Sampling to Sequencing and Analysis Christopher Quince1,^, Alan W
    Shotgun metagenomics, from sampling to sequencing and analysis Christopher Quince1,^, Alan W. Walker2,^, Jared T. Simpson3,4, Nicholas J. Loman5, Nicola Segata6,* 1 Warwick Medical School, University of Warwick, Warwick, UK. 2 Microbiology Group, The Rowett Institute, University of Aberdeen, Aberdeen, UK. 3 Ontario Institute for Cancer Research, Toronto, Canada 4 Department of Computer Science, University of Toronto, Toronto, Canada. 5 Institute for Microbiology and Infection, University of Birmingham, Birmingham, UK. 6 Centre for Integrative Biology, University of Trento, Trento, Italy. ^ These authors contributed equally * Corresponding author: Nicola Segata ([email protected]) Diverse microbial communities of bacteria, archaea, viruses and single-celled eukaryotes have crucial roles in the environment and human health. However, microbes are frequently difficult to culture in the laboratory, which can confound cataloging members and understanding how communities function. Cheap, high-throughput sequencing technologies and a suite of computational pipelines have been combined into shotgun metagenomics methods that have transformed microbiology. Still, computational approaches to overcome challenges that affect both assembly-based and mapping-based metagenomic profiling, particularly of high-complexity samples, or environments containing organisms with limited similarity to sequenced genomes, are needed. Understanding the functions and characterizing specific strains of these communities offer biotechnological promise in therapeutic discovery, or innovative ways to synthesize products using microbial factories, but can also pinpoint the contributions of microorganisms to planetary, animal and human health. Introduction High throughput sequencing approaches enable genomic analyses of ideally all microbes in a sample, not just those that are more amenable to cultivation. One such method, shotgun metagenomics, is the untargeted (“shotgun”) sequencing of all (“meta”) of the microbial genomes (“genomics”) present in a sample.
    [Show full text]
  • Human and Mouse Gene Structure: Comparative Analysis and Application to Exon Prediction
    Downloaded from genome.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Human and Mouse Gene Structure: Comparative Analysis and Application to Exon Prediction Serafim Batzoglou,1,4,7 Lior Pachter,2,7 Jill P. Mesirov,3 Bonnie Berger,1,4,6 and Eric S. Lander3,5,6 1Laboratory for Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA; 2Department of Mathematics, University of California Berkeley, Berkeley, California 94720 USA; 3Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 USA; 4Department of Mathematics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA; 5Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA We describe a novel analytical approach to gene recognition based on cross-species comparison. We first undertook a comparison of orthologous genomic loci from human and mouse, studying the extent of similarity in the number, size and sequence of exons and introns. We then developed an approach for recognizing genes within such orthologous regions by first aligning the regions using an iterative global alignment system and then identifying genes based on conservation of exonic features at aligned positions in both species. The alignment and gene recognition are performed by new programs called GLASS and ROSETTA, respectively. ROSETTA performed well at exact identification of coding exons in 117 orthologous pairs tested. A fundamental task in analyzing genomes is to identify by comparison of syntenic human and mouse genomic the genes. This is relatively straightforward for organ- sequences. isms with compact genomes (such as bacteria, yeast, It is well known that cross-species sequence com- flies and worms) because exons tend to be large and the parison can help highlight important functional ele- introns are either non-existent or tend to be short.
    [Show full text]
  • Eric S. Lander
    Chancellor's Distinguished Fellows Program 2004-2005 Selective Bibliography UC Irvine Libraries Eric S. Lander February 25, 2005 Prepared by: John E. Sisson Biological Sciences Librarian [email protected] Journal Articles (Selected from over 220 articles he has authored or co-authored) Poirier, C., Y. J. Qin, C.P. Adams, Y. Anaya, J. B. Singer, A. E. Hill, Eric S. Lander, J.H. Nadeau, and C. E. Bishop. "A complex interaction of imprinted and maternal-effect genes modifies sex determination in odd sex (Ods) mice." Genetics 168, no. 3 (2004): 1557-1562. Skuse, D. H., S. Purcell, M. J. Daly, R. J. Dolan, J. S. Morris, K. Lawrence, Eric S. Lander, and P. Sklar. "What can studies on Turner syndrome tell us about the role of X- linked genes in social cognition?." American Journal of Medical Genetics Part B- Neuropsychiatric Genetics 130B, no. 1 (2004): 8-9. Rioux, J. D., H. Karinen, K. Kocher, S. G. McMahon, P. Karkkainen, E. Janatuinen, M. Heikkinen, R. Julkunen, J. Pihlajamaki, A. Naukkarinen, V. M. Kosma, M. J. Daly, Eric S. Lander, and M. Laakso. "Genomewide search and association studies in a Finnish celiac disease population: Identification of a novel locus and replication of the HLA and CTLA4 loci." American Journal of Medical Genetics Part A 130A, no. 4 (2004): 345- 350. Michalkiewicz, M., T. Michalkiewicz, R. A. Ettinger, E. A. Rutledge, J. M. Fuller, D. H. Moralejo, B. Van Yserloo, A. J. MacMurray, A. E. Kwitek, H. J. Jacob, Eric S. Lander, and A. Lernmark. "Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat." Physiological Genomics 19, no.
    [Show full text]
  • Random Shotgun Fire
    Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press EDITORIAL Random Shotgun Fire Craig Venter’s and Perkin-Elmer’s May 9th announce- lengths. The sequencing machine will be accompanied ment of a new joint venture to complete the sequence by an automated workstation that does colony picking, of the human genome in just 3 years set off a furor extraction, PCR, and sequencing reactions. The com- among the scientific community. The uproar, how- pany is committed to publicly releasing data on a quar- ever, was unsurprising given that the earliest newspa- terly basis on contigs greater than 2 kb, though the per articles presented the plan as if it were a fait accom- exact details for this release are still under discussion. pli and accused the publicly funded Human Genome The general business plan of the company includes the Project of being a ‘‘waste’’ of money. The announce- intention to patent between 100 and 300 interesting ment, made just prior to the Genome Mapping, Se- gene systems. Additionally, they plan to position quencing, and Biology Meeting, held May 13–17 at themselves as a supplier of a sequence database and Cold Spring Harbor Laboratory, was discussed, at least analysis tools. Finally, they intend to exploit the dis- briefly, at the sequencing center director’s meeting covered single nucleotide polymorphisms (SNPs), that preceded the CSHL meeting, in a very well- probably through a new genotyping service. With attended session during the CSHL meeting, and in nu- their approach, they claim they can sequence the hu- merous speculative debates over meals and beers man genome in just 3 years at a cost of roughly $200 among attending scientists.
    [Show full text]
  • Whole Genome Shotgun Sequencing
    Whole Genome Shotgun Sequencing Before we begin, some thought experiments: Thought experiment: How big are genomes of phages, Bacteria, Archaea, Eu- karya?1 Thought experiment: Suppose that you want to sequence the genome of a bac- teria that is 1,000,000 bp and you are using a sequencing technology that reads 1,000 bp at a time. What is the least number of reads you could use to sequence that genome?2 Thought experiment: The human genome was “finished” in 2000 — to put that in perspective President Bill Clinton and Prime Minister Tony Blair made the announcement! — How many gaps remain in the human genome?3 A long time ago, when sequencing was expensive, these questions kept people awake. For example, Lander and Waterman published a paper describing the number of clones that need to be mapped (sequenced) to achieve representative coverage of the genome. Part of this theoretical paper is to discuss how many clones would be needed to cover the whole genome. In those days, the clones were broken down into smaller fragments, and so on and so on, and then the fewest possible fragments sequenced. Because the order of those clones was known (from genetics and restriction mapping), it was easy to put them back together. In 1995, a breakthrough paper was published in Science which the whole genome was just randomly sheared, lots and lots of fragments sequenced, and then big (or big for the time — your cell phone is probably computationally more powerful!) computers used to assemble the genome. This breakthrough really unleashed the genomics era, and opened the door for genome sequencing including the data that we are going to discuss here! As we discussed in the databases class, the NCBI GenBank database is a central repository for all the microbial genomes.
    [Show full text]