Overview of DNA Sequencing Strategies

Shendure, Jay; Porreca, Gregory J.; Church, George M.; Gardner, Andrew F.; Hendrickson, Cynthia L.; Kieleczawa, Jan; Slatko, Barton E.

doi:10.1002/0471142727.mb0701s96

Cited by 95 publications

(75 citation statements)

References 116 publications

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“…Illumina sequencing allows whole-genome sequencing utilizing clonal amplification of DNA fragments and sequence-by-synthesis approaches (48). While the method is highly accurate, fast, and relatively inexpensive, the Illumina platform yields short read lengths of between 50 and 300 bp, creating a major difficulty with respect to de novo genome assembly through repetitive genomic regions.…”

Section: Single-molecule Real-time Sequencingmentioning

confidence: 99%

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

et al. 2017

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Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remain a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Here, we discuss the analysis of data from transposon mutagenesis followed by deep sequencing (Tnseq) to determine gene disruptions differentially represented in a mutant population and Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real-time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications.KEYWORDS High-throughput, SMRT, Tn-seq, sequencing M any important advances in sequencing technologies can be attributed to studies in microbiology. The first sequenced genome of bacteriophage X174 was completed by Sanger in 1978 (1), followed by the shotgun sequencing of bacteriophage lambda (2) and then the 1.8-MB genome sequence for Haemophilus influenzae in 1995 (3). Subsequent efforts led to the development of next-and third-generation sequencing platforms (4). Recent advances have provided powerful tools for novel research in basic and translational bacteriology. Given the diversity and complexity of sequencing applications, the initial implementation of microbial genomics and subsequent data analysis may prove arduous for researchers new to genomics.In our experience, many laboratories unfamiliar with sequencing approaches or subsequent data analysis have become interested in implementing sequencing to enrich discovery in their studies. This minireview is not intended to be comprehensive or written for an expert. The goal of this minireview is to provide an introductory explanation, tools, and resources for bacteriologists new to sequencing approaches. Therefore, we have chosen to discuss the application of three popular sequencing approaches followed by highlighting a few examples from the literature. First, we discuss methods for assessing fitness subsequent to transposon mutagenesis followed by deep sequencing (Tn-seq). Tn-seq analysis can be used as a genome-wide gene discovery method in a broad range of microorganisms. Next, we discuss the bioinformatics tools available for applications in resequencing utilizing high-throughput sequencing. One application of resequencing is to rapidly identify suppressor mutations

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Section: Single-molecule Real-time Sequencingmentioning

confidence: 99%

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

et al. 2017

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“…Subsequently denaturation and cleanup of free moving enzyme, primers and the nucleotides, while the resulting molecules are arranged by their molecular weight (analogous of the point of termination) and the markers involved to the terminating ddNTPs is read out successively in order formed by the categorization step. With the application of existing Sanger sequencing techniques, it is precisely capable of 384 sequences [23] ranging from 600 to 1,000 nt, in length [24,25] though, the present 384-capillary systems are exceptional. The additional ordinary 96-capillary apparatus produce a utmost of more or less 6 Mb of DNA sequence apiece date, with expenses for consumables cost about $500/1 Mb.…”

Section: Key Hts Platforms and Applicationsmentioning

confidence: 99%

High Throughput Sequencing Advances and Future Challenges

Pervaiz¹,

Lotfi²,

Haider³

et al. 2017

J Plant Biochem Physiol

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High throughput sequencing (HTS) technologies were developed into indispensable for genomic investigation and recent hottest topic for research in the field of genomics, which can generate over 100 times more data in comparison with the most complicated capillary sequencers. Recent advances and developments in HTS using next generation sequencing techniques have become essential in the studies of digital gene expression profiling, in epigenomics, genomics, and transcriptomics. These methodologies are dexterous of sequencing multiple DNA molecules in corresponding; facilitate hundreds of millions of DNA molecules to be sequenced within a short period of time. Though, the expenses and time period have been significantly reduced; the inaccurate profiles and boundaries of the new policy differ considerably from those of earlier reported sequencing techniques. The technical developments and decreasing cost of NGS (Next Generation Sequencing) technology have made RNA sequencing (RNA-seq) as a worldwide popular technique for gene expression projects. Various approaches have been done for the standardization of RNA sequencing data, which have been materialized in the reports, contradictory, both in the type of bias modification and in the statistical approach. On the other hand, as data persistently build up, there has been no apparent consensus on the proper normalization techniques to be used or the impact of chosen methods on the downstream analysis. In the present article, we mentioned the key features of HT-NGS like, Key HTS platforms and different sequencing applications, ethical limitation and future prospective.

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“…Unfortunately, tools for analysis of minute quantities of DNA are currently inadequate or technically challenging. For example, advances in sequencing technology (i.e., nanopore sequencing) can use extremely long DNA but methods to create long DNAs have not kept pace (Branton et al, 2008;Metzker, 2010;Shendure et al, 2011). Novel amplification techniques are also required to profile genetic variations among single cells (Navin and Hicks, 2011;Schubert, 2011) because the quantity of genomic DNA from a single cell is insufficient to sequence directly.…”

Section: Future Challengesmentioning

confidence: 99%

DNA polymerases in Biotechnology

2015

Frontiers Research Topics

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Overview of DNA Sequencing Strategies

Cited by 95 publications

References 116 publications

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

High Throughput Sequencing Advances and Future Challenges

DNA polymerases in Biotechnology

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