Amplified Voices

At the UCSF Center for Advanced Technology (CAT), Dr. Chow and his team routinely process dozens to hundreds of research and surveillance samples, many of which have highly variable input quality and concentration. In workflows like viral sequencing, the standard approach relies on long, fixed-cycle PCR steps designed to average out these differences, oftentimes resulting in unnecessary over-amplification, hidden primer dimers, wasted sequencing capacity, and slow turnaround times.

Read the case study to see how Dr. Chow weighs the potential of iconPCR as an alternative mode of amplification control to make sequencing workflows cheaper, better, and faster.

The Center for Advanced Technology (CAT) at UCSF is at the forefront of providing cutting-edge sequencing services and access to technology for researchers. In a recent interview, Dr. Chow shared insights into the facility's operations, the challenges they faced in high-throughput sequencing, and how adopting iconPCR has significantly improved their workflow.

The bacterial 16S rRNA gene is widely used to characterize host-associated and environmental microbiomes, most commonly through sequencing short hypervariable regions. Recent improvements in PacBio sequencing chemistry and concatenation approaches can now enable high-throughput, full-length 16S rRNA gene sequencing with high accuracy and depth. However, PCR-induced errors remain a major limitation, particularly for full-length amplicons where error accumulation may be elevated due to a five-fold increase in sequence lengths. These challenges are exacerbated when samples vary widely in microbial biomass, making it difficult to select a single optimal number of PCR cycles.

This publication evaluates the effects of PCR cycle autonormalization on PacBio Kinnex full-length 16S rRNA gene sequencing across seven agriculturally relevant specimen types, comparing conventional PCR protocols with an autonormalization approach in which individual reactions were terminated during exponential amplification based on real-time fluorescence thresholds.

Insects have interconnected relationships with gut microorganisms, which can have impacts on behavior and survival. Understanding foundational ecological principles of the insect-associated microorganisms is important to understand how insects utilize microbes to cope with stress and different environments. Common microbiome surveys of insect microbiomes utilize 16S rRNA sequencing of metagenomic DNA. For amplicon-based surveys of insect microbiome, fragment length and 16S rRNA subunit choice may have unintended biases, and some primer combinations may under-represent genus or species richness.

This preprint illustrates the benefit of full-length 16S rRNA sequencing in improving sample resolution compared to V4, which provides new insight into the gut microbiome community composition of an invasive insect and reveals substantial differences in bacterial composition across different medfly cohorts when sampling the full-length amplicons.

Cell-free DNA (cfDNA) sequencing is one of the most demanding workflows in genomics. Whether used for minimal residual disease (MRD) monitoring, tumor evolution tracking, or early cancer detection, cfDNA applications are fundamentally limited in their initial material: inputs are scarce, samples are precious, and every lost molecule represents a potential missed signal.

Read this application note to see how the team used icon96 to generate uniform library yields across highly variable inputs — without sacrificing data quality or requiring labor-intensive upfront quantification.

The purpose of this study was to evaluate the use of carrier DNA for shotgun metagenome sequencing of ultra-low levels of metagenomic DNA. The team hypothesized that carrier DNA would improve the robustness of library preparation for samples with DNA concentrations below detection by providing a tangible amount of known DNA, thereby bringing total DNA concentrations closer to recommended input ranges for metagenomic library kits.

Adaptive PCR cycling using an iconPCR instrument was employed to allow dynamic thermocycling until sufficient library for sequencing was amplified, regardless of input DNA concentration. Adaptive cycling allowed appropriate cycling conditions when input DNA concentrations were below detection.

Standard PCR systems require users to set a predetermined number of cycles, based on assay and sample-specific factors. For single cell sequencing experiments, this requires knowledge of how many cells are captured and the RNA content within the cells. Different cell numbers and RNA quality require careful curation of the PCR cycle numbers for each sample, necessitating multiple thermocycler runs, which increases workflow complexity and potential sources of error.

icon96 unlocks new single cell workflows, allowing for samples of different types and inputs to be run simultaneously, increasing efficiency, preventing erroneous cycling conditions, and allowing for a simplified workflow — while maintaining the quality of the single cell gene expression data.