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

ABSTRACT: We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.

SUBMITTER: Adey A

PROVIDER: S-EPMC3046479 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Adey Andrew A Morrison Hilary G HG Asan Xun Xu X Kitzman Jacob O JO Turner Emily H EH Stackhouse Bethany B MacKenzie Alexandra P AP Caruccio Nicholas C NC Zhang Xiuqing X Shendure Jay J

Genome biology 20101208 12

We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library constr ...[more]

PMID: 21143862

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

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

| PRJNA274948 | ENA

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

Project description:BackgroundConstruction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs.ResultsWe report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases.ConclusionsA simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

| S-EPMC4170940 | biostudies-literature

Low-input Hi-C library construction

Project description:To evaluate the robustness of CtG, we constructed Hi-C libraries of varying quantities of cell inputs utilizing a low-input Hi-C technique.

2023-07-19 | GSE233166 | GEO

Construction of Reusable Plasmid Libraries for Strain Construction

Project description:Test whether it is possible to conjugate a whole plasmid library into a recipient strain without loss of fidelity (as judged by aCGH analysis) Donor and Recipient Libraries are compared with array CGH to detect copy number differences, a simple selection experiment is also performed

2013-12-31 | E-GEOD-44258 | biostudies-arrayexpress

Construction of Reusable Plasmid Libraries for Strain Construction

Project description:Test whether it is possible to conjugate a whole plasmid library into a recipient strain without loss of fidelity (as judged by aCGH analysis)

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Low-cost, low-bias and low-input RNA-seq with High Experimental Verifiability based on Semiconductor Sequencing

Project description:We developed a new method on sequencing low-input RNA. This method shows much low-bias with the advantage of semiconductor while competing with smart-seq2. In order to analyze the low-input RNA datasets sensitively, we also develop FANSe2splice with high experimental verification rate as the analysis tool in our method.

2017-06-12 | GSE87660 | GEO

Low-cost, Low-bias and Low-input RNA-seq with High Experimental Verifiability based on Semiconductor Sequencing.

Project description:Low-input RNA-seq is powerful to represent the gene expression profiles with limited number of cells, especially when single-cell variations are not the aim. However, pre-amplification-based and molecule index-based library construction methods boost bias or require higher throughput. Here we demonstrate a simple, low-cost, low-bias and low-input RNA-seq with ion torrent semiconductor sequencing (LIEA RNA-seq). We also developed highly accurate and error-tolerant spliced mapping algorithm FANSe2splice to accurately map the single-ended reads to the reference genome with better experimental verifiability than the previous spliced mappers. Combining the experimental and computational advancements, our solution is comparable with the bulk mRNA-seq in quantification, reliably detects splice junctions and minimizes the bias with much less mappable reads.

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CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples.

Project description:Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.

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Construction of high-density genetic linkage maps for orange-spotted grouper Epinephelus coioides using multiplexed shotgun genotyping.

Project description:BACKGROUND: Orange-spotted grouper, Epinephelus coioides, is one of the most valuable fish species in China. Commercial production of orange-spotted grouper could be increased by developing higher growth rates and improving commercially important traits. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. A high-density genetic linkage map is the basis for QTL study, and multiplexed shotgun genotyping (MSG) facilitates the development of single nucleotide polymorphisms (SNPs) and genotyping. In this study, the first high-density genetic linkage maps for groupers were generated on the basis of the MSG method. RESULTS: The sex-averaged map contained a total of 4,608 SNPs, which spanned 1581.7 cM, with a mean distance between SNPs of 0.34 cM. The 4,608 SNPs were located in 2,849 unique locations on the linkage map, with an average inter-location space at 0.56 cM. There were 2,516 SNPs on the female map, and the number of unique locus was 1,902. However, the male map contained more numbers of SNP (2,939) and unique locations (2,005). The total length of the female and male maps was 1,370.9 and 1,335.5 cM, respectively. CONCLUSIONS: The high-resolution genetic linkage maps will be very useful for QTL analyses and marker-assisted selection (MAS) for economically important traits in molecular breeding of the orange-spotted grouper.

| S-EPMC3890575 | biostudies-literature

Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.

Project description:Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.

| S-EPMC3209006 | biostudies-literature

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