Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

Adey, Andrew; Morrison, Hilary G.; Asan,; Xu, Xun; Kitzman, Jacob O.; Turner, Emily H.; Stackhouse, Bethany; MacKenzie, Alexandra P.; Caruccio, Nicholas; Zhang, Xiuqing; Shendure, Jay

doi:10.1186/gb-2010-11-12-r119

Cited by 518 publications

(525 citation statements)

References 23 publications

Supporting

Mentioning

503

Contrasting

Order By: Relevance

“…4); this trait was not observed when using the sonication method. As previously reported, this ''Nextera'' signature indicates that the transposon insertion is not completely random (Adey et al, 2010). However, this bias seems negligible as far as the gene expression profile was concerned, which strongly supports the previous studies (Adey et al, 2010;Gertz et al, 2012).…”

Section: Resultssupporting

confidence: 89%

“…As previously reported, this ''Nextera'' signature indicates that the transposon insertion is not completely random (Adey et al, 2010). However, this bias seems negligible as far as the gene expression profile was concerned, which strongly supports the previous studies (Adey et al, 2010;Gertz et al, 2012). Using oneway analysis of variance (ANOVA) and filtering based on a two-fold change up/down with an FDR-corrected P-value of 0.05 we observed only three genes as significantly changed in expression profile between the library preparation methods.…”

Section: Resultssupporting

confidence: 76%

“…When the fragmentation and the tag-addition are finished, 9 bp of gap in the nontransferred strand is created due to the action of the transposase (Reznikoff, 2008;Adey et al, 2010;Gertz et al, 2012). After this gap is filled, the library is constructed with PCR of minimum cycles.…”

Section: Developmental Dynamicsmentioning

confidence: 99%

“…Furthermore, sample loss at each step increases the amount of starting material required, something which is likely a limiting factor in single-cell analysis. On the other hand, library construction for deep sequencing with hyperactive Tn5 transposase has been shown to provide high quality libraries from genomic DNA as well as RNA-seq; the protocol offers simplicity and reproducibly with fewer steps (Adey et al, 2010;Gertz et al, 2012). This hyperactive Tn5 transposase is bound to synthetic DNA, which contains 19 bp of the ''mosaic end-recognition sequence'' as well as the flanked adaptors for deep sequencing, to form the transposase complex ( Fig.…”

Section: Introductionmentioning

confidence: 99%

“…This hyperactive Tn5 transposase is bound to synthetic DNA, which contains 19 bp of the ''mosaic end-recognition sequence'' as well as the flanked adaptors for deep sequencing, to form the transposase complex ( Fig. 2b; Adey et al, 2010;Gertz et al, 2012). The mosaic endrecognition sequence, which is a tandem conjugate sequence of outsideend and inside-end recognition sequences in the insertion sequence of the wild-type Tn5 transposon, is necessary for random integration into the target DNA.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

Brouilette

Kuersten

Mein

et al. 2012

Developmental Dynamics

View full text Add to dashboard Cite

Background: Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Results and Conclusions: Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Developmental Dynamics 241:1584-1590, 2012. V C 2012 Wiley Periodicals Inc.

show abstract

Section: Resultssupporting

confidence: 89%

Section: Resultssupporting

confidence: 76%

Section: Developmental Dynamicsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

Brouilette

Kuersten

Mein

et al. 2012

Developmental Dynamics

View full text Add to dashboard Cite

show abstract

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

Ellington²,

et al. 2013

View full text Add to dashboard Cite

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance.

show abstract

Exome Sequencing by Targeted Enrichment

Clark

Chen

Snyder

2013

CP Molecular Biology

View full text Add to dashboard Cite

This unit describes methods for targeted enrichment of the exon-coding portions of the genome using Agilent SureSelect Human All Exon 50 Mb and Roche Nimblegen SeqCap EZ Exome platforms. Each platform targets and enriches a large overlapping portion of the greater human exome. The protocols here describe the biochemical procedures used to enrich exomic DNA with each platform, including recommended modifications to the manufacturers' protocols. In addition, a brief description of the sequencing protocol and estimation of the needed amount of sequencing for each platform is included. Finally, a detailed analytical pipeline for processing the subsequent data is described. These protocols focus specifically on human exome sequencing platforms, but can be applied with some modification to other organisms and targeted enrichment approaches.

show abstract

Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

Cited by 518 publications

References 23 publications

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

A simple and novel method for RNA‐seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

Exome Sequencing by Targeted Enrichment

Contact Info

Product

Resources

About