Titration-free massively parallel pyrosequencing using trace amounts of starting material

Abstract

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable.

Figure 1.

Schematic description of three types of library construction.

Figure 2.

(A) Enrichment percentage, single copy and mixed copy DNAs in an emulsion drop/bead according to Poisson theory. In a subset (B), enriched percentage as a function of input DNA-to-bead ratio approaches linearity when DNA-to-bead ratio decreases. The 8% enrichment percentage recommended by the Roche 454 protocol corresponds to a 0.083 DNA-to-bead ratio. (C) Sequencing outcome metrics as functions of the percentage of DNA-bearing beads (called enrichment percentage if enrichment were performed). The 40–60% enrichment percentage zone harbored a critical turning point, after which undesired beads becoming dominant quickly. As indicated by asterisks the Roche 454 Dot filter filters away those reads having too many negative flows due to poor incorporations or interruptions, the ShortQuality filter filters those reads failed the length test because of quality trimming and the ShortPrimer filter filters those failed the length test because of primer sequence trimming. As indicated by dagger the Roche 454 pipeline uses a positive flow percentage of 70% as a cut-off to distinguish Single- from Mixed-copy of templates.

Figure 3.

Nine stock libraries were previously quantified by BioAnalyzer as described in the Roche 454 Manual (A) and re-quantified by qPCR (B). Each colour indicates one library with four titration points. The blue curve indicates Poisson prediction of enrichment percentage. There was a deviation from Poisson prediction in (A) (P = 0.0005) and no deviation in (B) (P = 0.9157).

Figure 4.

Library quantification by qPCR. All samples were run in triplicate and only the ones with median Ct values were plotted. (A) The six standards ranged from 10⁷ to 10² copies. The tested AB library was 38 200 molecules/µl. The reactions containing the AB library template and one of the emPCR primers (A only or B only) generated no detectable fluoresces. (B) Gel electrophoresis of qPCR products. Lane 1: standard 10⁴ copies; lane 2: no template control; lane 3: tested AB library; lane 4: the reaction containing the library template and primer A only; lane 5: the reaction containing the library template and primer B only. (C) The standards ranged from 10⁷ to 10⁴ copies. The tested Y library was 894 000 molecules/µl. (D) Gel electrophoresis of qPCR products. Lane 1: no template control; lane 2: Y library.

Figure 5.

Enrichment percentage observed from emulsion-titration assay and predicted by Poisson distribution.