A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing - PubMed (original) (raw)

A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing

Poornima Parameswaran et al. Nucleic Acids Res. 2007.

Abstract

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward-reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n(2) independent libraries for each set of 'n' forward and 'n' reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.

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Figures

Figure 1.

Figure 1.

Design of forward and reverse primers. The synthesized primers are 45–46 nt long. (A and B) The template for the primers is: 454-Adapter:: Barcode:: Linker-Primer. Individual specifications for forward and reverse barcodes are also indicated. (C) Diagrammatic representation of the GS20 forward and reverse sequencing reads. ‘RC’ stands for Reverse Complement. Teeth denote base pairing.

Figure 2.

Figure 2.

A diagrammatic representation of heteroduplexes that may be formed in an amplified pool. (A) Heteroduplexes formed between molecules from the same sample that have the same barcode, but different RNA inserts (X and Y). (B) Heteroduplexes formed between molecules with RNA inserts and molecules with no RNA inserts (or with fragments of linkers as inserts). Formation of these unusual duplexes may be facilitated by the 45–46 nt complementarity at either end of the insert. Thus, three types of molecules may be present during later stages of PCR: single-stranded, perfectly double stranded (Figure 1C) and heteroduplexes (shown here). The ratio of the three species is determined by the number of PCR cycles. As in Figure 1, ‘RC’ stands for Reverse Complement, and teeth denote base pairing.

Figure 3.

Figure 3.

Visualizing the nature of amplified products from various cycles of PCR. With an increase in cycle number (twenty cycles of an initial round of PCR followed by six, eight or ten cycles of a second round of PCR), there is an evident shift in mobility of PCR products that contain a small RNA insert. Effect of PCR cycle number on three different samples is shown, stressing the importance of titrating the total number of DNA amplification cycles, to avoid saturation of the PCR amplification. Red arrows represent the two sizes of the insert-containing PCR products. PCR products without small RNA inserts migrate as faint bands between 75 and 100 bp (black arrow).

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