Absolute quantification of NGS libraries with digital PCR lowers sequencing workflow costs
Next-generation sequencing (NGS) libraries can be quantified with minimal sample handling and without the need to generate a standard curve using digital PCR. This method enables accurate and precise library quantification, a critical step in both the Ion Torrent™ and Illumina® workflows, allowing for maximizing sequencing yields downstream. To achieve this high degree of precision, a TaqMan® Assay, designed to span both the forward and reverse adapters specific to each library, is available.
This approach limits quantification to library constructs that contain both adapter sequences. Ultimately, using digital PCR to quantify NGS libraries decreases overall sequencing costs by ensuring an accurate quantification upfront, minimizing the need to re-run or repeat sequencing of samples.
Figure 1. Schematic of TaqMan® assay design for NGS library quantification on the QuantStudio™ 3D Digital PCR system. The assay contains a forward primer, reverse primer, and TaqMan® probe all complementary to the Ion Torrent™ or Illumina® library adapter sequences.
Figure 2. Calculated copies/µL of various library types by QuantStudio™ AnalysisSuite™ software. The software takes into account the dilution factor used when defining chips. Error bars indicate 95% a confidence level for each sample. The red oval indicates replicate chips for samples L787.
||Library type (size)
||Concentration determined by digital PCR
||Templated beads pre-enrichment
||Fragment (276 bp)
Fragment (489 bp)
||Fragment (322 bp)
Table 1: Correlation of QuantStudio™ 3D Digital PCR data with that of the Ion OneTouch™ 2 data. These quality-control data were generated for four Ion Torrent™ libraries using flow cytometry prior to enrichment.
Figure 3. Alternative views in the Absolute Quantification application module of the QuantStudio™ 3D AnalysisSuite™ software of the small RNA (A) and Nextera (B) libraries. Example of a chip view depicting color by calls, which shows the distribution of both the VIC (amplified) and the non-amplified wells. Note that a random distribution of VIC signal across the chip is ideal. The histogram view has two populations, the larger yellow population corresponds to the non-amplified wells with a lower fluorescence and the smaller red population corresponds to the amplified wells with a significantly higher fluorescence. The separation between non-amplified and amplified populations is indicative of good discrimination.
Figure 4. Correlation of QuantStudio™ 3D Digital PCR data with that of the Illumina® cluster density data. (A) Graph depicting the concentration (nM) for five Illumina® libraries as determined by the QuantStudio™ 3D Digital PCR System. (B) Cluster densities for the same libraries generated by the HiSeq. Library 2 shows under clustering as is evident by the lower concentration as calcuated by dPCR.