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Encore™ Library Spike-in Controls
The Encore Library Spike-In Controls are designed to be used as an internal positive control that enables qualitative assessment of the library construction process before and after sequencing. This facilitates (or something more active) an assessment of the library construction procedure independent of the sample quality and quantity. In addition, this process complements other quantitative library QC procedures, such as yield, and is broadly applicable to all next-generation sequencing sample types and platforms.
The Library Spike Mix contains a cocktail of three unphosphorylated, double-stranded DNA fragments approximately 200 bp in length with sequences selected from a deep sea vent bacterium. These sequences show minimal homology to commonly sequenced species. The concentrations of the spike-ins are staggered spanning two orders of magnitude.
The Library Spike Mix is added to double-stranded, DNA samples prior to library construction with the NuGEN Encore NGS Library Systems, or any other commercial library preparation methods. A library is then constructed using the spike +sample mixture; including end repair, ligation of adaptors, and PCR amplification. The three sets of qPCR primers provided with the kit are designed to quantitate the spike-ins after the library is prepared to assess the efficiency of the library construction process independent from the samples.
Figure 1. Example library yields and spike-in qPCR Ct values for various input sample types and sample or library construction failure modes.
Artificially introduced library failure modes were analyzed by library yield, Bioanalyzer traces of the libraries and qPCR of the spikes. Three different input materials (E.coli genomic DNA, NuGEN Ovation® RNA-Seq samples and Human gDNA) were used as input for the Encore Library Multiplex System. The control samples (Ctrl) were prepared following the recommended procedure while three library processing failure modes were tested including No ER (no end repair), No Lig (no ligation), or No Amp (no PCR amplification). As shown in Figure 1, all control samples had expected high yield (above 1 µg) with all three spike-ins detectable and in the expected order (Spike 1 < Spike 2 < Spike 3). In contrast, the test samples intentionally failing one of the library preparation steps resulted in low yield (< 0.5 µg) and associated with extremely high Ct values (above 28), consistent with failed library process.
The two sample negative controls, highly degraded FFPE Liver gDNA and Negative Ctrl (0 ng input), produced different patterns where low library yields were produced while positive spike-in controls gave expected Ct values. This combination is diagnostic of poor input sample quality although library construction proceeded successfully.
Note that the Encore Library Spike-in Controls are compatible with all Ovation amplification kits with the exception of the Ovation WGA FFPE System, which uses a different adaptor ligation strategy.
Table 1. Sequencing counts data from libraries with and without spike-in controls
The performance of the spike-in controls can also be tracked after sequencing. However, applications that utilize a target enrichment strategy may not sufficiently enrich for spike-ins resulting in lower or absent counts in the final sequencing analysis.