NGS library normalization is a labor-intensive process that uses costly reagents. iconPCR’s AutoNormalization^TM enables customers to combine quantification and normalized pooling into one step to produce high quality libraries by ensuring individual reactions are amplified to the same levels. With iconPCR, libraries can be pooled prior to SPRI-bead cleanup, for a simple one-step single-tube purification, drastically reducing the most labor-intensive portion of the workflow and manual errors.
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Whole genome libraries were generated using NA12878 DNA across a 120-fold dilution series of 20, 10, 5, 2.5, 1.25 and 0.625 nanograms per reaction. iconPCR auto-normalized samples were pooled into a single SPRI-bead clean-up. Conventional PCR with a fixed number of cycles was performed on the same series. Here we show the significant yield variance of the fixed PCR protocol versus iconPCR’s of all reactions amplifying to within 1 Cq.
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• >24 fewer touch points
• Workflow reduced by 60% (for 96 samples)
• >90% reduction in consumables

Metagenomics sample variability in complexity and quality necessitates more amplification cycles for small amounts of material, risking chimeras and concatemers that lower sequence quality and yield. Overamplification from traditional PCR can skew species representation, diminishing actual diversity and leading to false negatives.

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Data showing the species representation as identified by full length 16S sequencing from 32 different soil samples. Top image: species distribution in libraries generated using the conventional PCR. Bottom image: libraries generated using iconPCR AutoNormalization.

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• Quasi-removal of chimeras and concatemers
• Several fold increase in unique sequences
• Accurate representation of the population diversity

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FFPE samples, notorious for poor quality, see significant improvement with iconPCR. Using AutoNormalization library concentrations are normalized, reducing amplification challenges and adapter dimers. IconPCR streamlines FFPE workflows, reducing failures and unnecessary sequencing. This method rescues samples often lost with conventional PCR and ensures more dependable results and higher yields.

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Comparison of FFPE libraries prepared using the conventional method and iconPCR’s AutoNorm. Samples 2-5 were of extremely poor quality (see DIN values),
but were rescued by iconPCR. Medium quality libraries showed higher yields vs. conventional PCR.

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• Rescue poor quality samples
• Superior genomic coverage
• Avoid PCR artifacts

One of the key challenges in single-cell genomics lies in the library preparation process. With a complex protocol and multiple amplification steps, the success of single cell studies hinges on correctly assessing the quality of the starting material. iconPCR enables customers to bypass initial quantification and final normalization steps, producing high quality libraries with a much simpler workflow.
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Comparison of 10X 3’ scRNA outcomes using traditional methods (Control) versus iconPCR shows the profiles are virtually identical, but iconPCR offers a significantly simpler workflow.

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• Significant workflow streamlining
• Bypass starting material assessment
• Limit loss of biological sample

In spatial transcriptomics, library preparation is characterized by its lengthy and intricate nature, heavily reliant on the accurate evaluation of starting material quality. iconPCR offers a solution by allowing users to skip the initial quantification and final normalization steps, streamlining the process to create high-quality libraries with ease.

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Comparison of Visium results obtained by conventional method (Control) vs iconPCR. The spatial profiles are indistinguishable between the two methods.

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• Significant workflow streamlining
• Precise control of cDNA amplification
• Limit loss of biological sample

RNA sequencing (RNA-Seq) has emerged as a powerful and widely adopted technology for studying gene expression, allowing for detection of alternative splicing events and novel transcripts. However, despite its widespread use, challenges persist, particularly in the critical step of library preparation. RNA-Seq libraries are hampered by complex protocols that are susceptible to biases, RNA quality issues and variability. iconPCR automated process streamlines library preparation and produces more consistent samples for RNA-seq.

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Overamplification in human brain mRNA RNA-seq libraries leads to reduced gene counts and biases. The trend demonstrates the loss of useful sequences as the number of cycles passes an optimal number.

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• Workflow reduced by up to 40%
• Enhancement of the number of useful sequences
• Reduction of PCR duplicates