Titration-free 454 sequencing using Y adapters

Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
Nature Protocol (Impact Factor: 9.67). 08/2011; 6(9):1367-76. DOI: 10.1038/nprot.2011.369
Source: PubMed


We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe-based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment. A single quality control step prior to emulsion PCR ensures that libraries contain no adapter dimers and have an optimal length distribution. The presented protocol takes ∼7 h to prepare eight barcoded libraries from genomic DNA into libraries that are ready to use for full-scale emPCR. It will be useful, for example, to allow analyses of precious clinical samples and amplification-free metatranscriptomics.

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Available from: Zongli Zheng, Nov 16, 2014
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    • "In 2008, next-generation sequencing techniques were established. We then submitted the PaP1 genome to the CNHGC (Shanghai, China) for sequencing with a Roche/454 GS FLX titanium system [12]. In brief, the purified genomic DNA of PaP1 was fragmented, ligated to adapters and separated into single strands; the DNA fragments were bound to beads and amplified by emulsion PCR. "
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    ABSTRACT: Background Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. Results After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. Conclusions This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-803) contains supplementary material, which is available to authorized users.
    BMC Genomics 09/2014; 15(1):803. DOI:10.1186/1471-2164-15-803 · 3.99 Impact Factor
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    • "To avoid this, two alternatives may work: using a MGB label that does not interfere with Roche Y adaptors, or synthesizing Y adaptors without the fluorochrome [26]. Thus, we used Y adaptors with MGB-Taqman probes designed by Zheng and collaborators, enabling us to quantify the exact number of amplifiable molecules of these minimal libraries by qPCR [26]. Once the exact number of amplifiable molecules is calculated, the user can proceed directly to proper emPCR without the need to perform the previous emPCR titration step, saving further DNA [23], [26], [29]. "
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    ABSTRACT: The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.
    PLoS ONE 06/2014; 9(6):e97379. DOI:10.1371/journal.pone.0097379 · 3.23 Impact Factor
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    • "DNA collected was then sheared by sonication using Bioruptor™ (Diagenode). 454™ libraries were prepared according to Zheng et al [17]. Sequencing was performed using an FLX Titanium platform. "
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    ABSTRACT: Metagenomics is the genomic study of uncultured environmental samples, which has been greatly facilitated by the advent of shotgun-sequencing technologies. One of the main focuses of metagenomics is the discovery of previously uncultured microorganisms, which makes the assignment of sequences to a particular taxon a challenge and a crucial step. Recently, several methods have been developed to perform this task, based on different methodologies such as sequence composition or sequence similarity. The sequence composition methods have the ability to completely assign the whole dataset. However, their use in metagenomics and the study of their performance with real data is limited. In this work, we assess the consistency of three different methods (BLAST + Lowest Common Ancestor, Phymm, and Naive Bayesian Classifier) in assigning real and simulated sequence reads. Both in real and in simulated data, BLAST + Lowest Common Ancestor (BLAST + LCA), Phymm, and Naive Bayesian Classifier consistently assign a larger number of reads in higher taxonomic levels than in lower levels. However, discrepancies increase at lower taxonomic levels. In simulated data, consistent assignments between all three methods showed greater precision than assignments based on Phymm or Bayesian Classifier alone, since the BLAST + LCA algorithm performed best. In addition, assignment consistency in real data increased with sequence read length, in agreement with previously published simulation results. The use and combination of different approaches is advisable to assign metagenomic reads. Although the sensitivity could be reduced, the reliability can be increased by using the reads consistently assigned to the same taxa by, at least, two methods, and by training the programs using all available information.
    BMC Bioinformatics 03/2014; 15(1):90. DOI:10.1186/1471-2105-15-90 · 2.58 Impact Factor
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