From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing - PubMed (original) (raw)
From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing
Matthias Meyer et al. Nucleic Acids Res. 2008 Jan.
Abstract
Current efforts to recover the Neandertal and mammoth genomes by 454 DNA sequencing demonstrate the sensitivity of this technology. However, routine 454 sequencing applications still require microgram quantities of initial material. This is due to a lack of effective methods for quantifying 454 sequencing libraries, necessitating expensive and labour-intensive procedures when sequencing ancient DNA and other poor DNA samples. Here we report a 454 sequencing library quantification method based on quantitative PCR that effectively eliminates these limitations. We estimated both the molecule numbers and the fragment size distributions in sequencing libraries derived from Neandertal DNA extracts, SAGE ditags and bonobo genomic DNA, obtaining optimal sequencing yields without performing any titration runs. Using this method, 454 sequencing can routinely be performed from as little as 50 pg of initial material without titration runs, thereby drastically reducing costs while increasing the scope of sample throughput and protocol development on the 454 platform. The method should also apply to Illumina/Solexa and ABI/SOLiD sequencing, and should therefore help to widen the accessibility of all three platforms.
Figures
Figure 1.
Overview scheme of 454 sequencing. Double-stranded sequencing templates are blunt end repaired, and two universal adapters, A and B, are ligated to their ends. The B-adapter carries a 5′ biotin (I). Streptavidin beads are used to isolate only molecules carrying an A and a B adapter (II). The single-stranded sequencing library is melted from the beads through alkaline treatment (III). A PCR reaction mix containing 600 000 oligonucleotide-coated sepharose beads and an appropriate number of library molecules is emulsified to produce physically separated droplets as reaction vessels (IV). After amplification, the emulsion is broken while the PCR products remain attached to the beads. Since most beads remain empty in emPCR, amplified beads are isolated through a bead enrichment procedure (V). A total of 50 000 beads are required for loading onto the wells of a 16th 454 FLX picotitre plate region, and the sequencing reaction is performed by flowing nucleotides over the plate and measuring light emissions. Beads carrying multiple amplicons produce mixed signals, which are recognized and filtered out by the run-processing software.
Figure 2.
Five sequencing libraries were re-quantified in parallel to show the performance of qPCR quantification for libraries derived from different types of initial material. (A) Amplification plots of the standard and the libraries are drawn in black and red, respectively, with duplicates being treated collectively. The bonobo genomic DNA library was measured in a 1:30 dilution to obtain a signal within the range of the standard curve. The library from pooled PCR products was measured in the working dilution (1:100) used for emulsion PCR. (B) qPCR amplicons were size fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining in order to estimate the mean fragment size for each library.
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