DeCodiFi™ Long&Complex

DeCodiFi™ Long&Complex establishes a superior library baseline from ultra low input (1 ng) by shifting fragment distribution toward higher molecular weights. Unlike other polymerases that produce standard smears or non-specific artifacts, Long&Complex delivers the highest concentration of clean, viable libraries required for high-fidelity genomic reconstruction.

Figure 1: Library amplification QC. Comparison of E. coli and S. epidermidis library yields using five polymerases: DeCodiFi™  Long&Complex (L&C), KOD Xtreme™ Hot Start DNA Polymerase (KX), Q5® Hot Start High-Fidelity DNA Polymerase (Q5), PrimeSTAR® GXL DNA Polymerase (GXL), and LongAmp® Taq DNA Polymerase (LA). Libraries were generated using the PacBio’s Ampli-Fi protocol with 1 ng of DNA input per 50 µL reaction and a standardized 14-cycle amplification program. Samples were analyzed via agarose gel electrophoresis to characterize size distribution and yield prior to PacBio sequencing.

DeCodiFi™ Long&Complex facilitates chromosome-level reconstruction in the PacBio® Ampli-Fi™ protocol by delivering the long HiFi reads necessary to resolve complex architectures. By maintaining high processivity without fragment length compression, it maximizes assembly contiguity in ultra-low input workflows.

E. coli sequencing libraries generated via the PacBio® Ampli-Fi™ protocol. Libraries were amplified using DeCodiFi™ Long&Complex and industry-standard polymerases from a 1 ng DNA input in 50 µL reactions over 14 cycles. Gene completeness was assessed using BUSCO with the enterobacterales_odb12 dataset for E. coli. To ensure compatibility, E. coli samples were normalized to a uniform depth of 63X, corresponding to the lowest coverage sample for the genome.

DeCodiFi™ Long&Complex optimizes Oxford Nanopore™ assembly by capturing ultra-long fragments up to 47.1 kb. By maximizing fragment reach, it delivers a 65% increase in Contig N50, enabling researchers to achieve superior assembly contiguity and efficiency in complex genomic architectures through optimized coverage and reduced sequencing costs.

DeCodiFi™ Long&Complex is optimized for challenging templates, including long DNA fragments up to 30 kb, high-GC regions up to 90% GC, low DNA inputs, and templates containing expansion repeats.

DeCodiFi™ Long&Complex is recommended for PCR amplification of high-GC or complex DNA regions, including regulatory elements and repetitive sequences. It is also designed to support library amplification for long-read sequencing workflows involving low-input samples, high-GC genomes, or long targets that require high fidelity and robust performance.

The 2X All-In-One Mix contains DeCodiFi™ Hot-Start High-Fidelity Polymerase, dNTPs, MgCl₂, stabilizers, and GC enhancers in a proprietary buffer optimized for higher coverage. Primers and template DNA are not included.

Yes. Due to the strong 3′-exonuclease proofreading activity of DeCodiFi™ Long&Complex, we recommend incorporating two phosphorothioate bonds at the 3′ ends of primers. This modification prevents primer degradation, improves specificity, and minimizes primer dimer formation.

Always set up reactions on ice to protect primers from exonuclease activity. Mix components in a sterile tube or plate, centrifuge briefly, and transfer immediately to the thermal cycler. For optimal results, we strongly recommend a temperature gradient (starting 4°C below the lowest calculated Tm​) to identify the ideal annealing stringency.

Template input should be calibrated based on genomic complexity. Aim for at least 104 copies per reaction. As a baseline for a 25 µL reaction:

• Prokaryotic genomes: 5–10 ng (e.g., >0.05 ng for E. coli).
• Eukaryotic genomes: 10–50 ng (e.g., >34 ng for Human gDNA).
• Plasmids or Phages: ≤1 ng.

Use a rate of 30 seconds per kilobase. The optimal temperature depends on your goal:

• 68°C: Recommended for library amplification or when maximizing yield is the priority.
• 72°C: Recommended for amplicon PCR to ensure higher specificity.

Note: For repetitive fragments or non-specific products, a step-down program is also advised.

Standard protocols use 10–30 cycles. We recommend using the minimum number of cycles required for your specific input; lower cycling reduces the probability of stochastic errors, non-specific products, and smearing.

Yes, a gradient PCR is strongly recommended when setting up cycling conditions. Use the lower primer Tm as a reference, starting 4°C below that value and increasing in 2°C increments up to 4°C above the calculated Tm.

For expanded repeats or nonspecific amplification in long amplicons, use the stepdown PCR program provided in the protocol. For expanded repeats with 100% GC-rich content, contact technical support for guidance on the most appropriate solution for your application.

Store DeCodiFi™ Long&Complex at -20°C upon arrival and avoid repeated freeze-thaw cycles. The mix is designed to be transported at 2–8°C without loss of performance for up to 7 days, helping reduce dry ice use and simplify logistics.

Yes. If you are working with long fragments, high-GC targets, repetitive regions, expansion repeats, or low-input samples, our team can help evaluate template input, primer design, cycling conditions, gradient setup, and whether a stepdown PCR strategy is appropriate.

In amplification-based long read sequencing workflows, the polymerase can influence read-length distribution, GC representation, and library compression. These factors can affect downstream assembly contiguity, especially when starting from ultra-low DNA input.