Sources of Typical Errors in Next-Generation Sequencing

The benefits are numerous, as it is possible to investigate complete genomes in a single NGS experiment and quickly uncover novel variations and transcripts.

FREMONT, CA:In next-generation sequencing operations, PCR cyclers and consumables are typical error contributors. The nightmare scenarios include nonspecific products, yield loss, and incorrect outcomes. Runs must repeat at the expense of material samples, time, and resources. They do not wish for PCR to become a trap in NGS. Next-generation sequencing (NGS) merits its designation as a "next-generation" method based on its discovery power, sensitivity, scalability, and throughput. Target regions, including substantial sections of genes, can sequence in many samples. Large-scale SNP discovery is possible. Massive parallel sequencing provides numerous potentials for addressing some biological issues.

PCR is the foundation of the majority of NGS techniques. Applications include converting samples to sequencing-ready amplicon libraries and enriching samples and libraries, including quality control and quantification. PCR is employed in whole genome sequencing and targeted sequencing library creation, as NGS experiments involve difficult and costly procedures. It is evident what matters in NGS PCR: Based on optimal resource management, successful sample 

processing and reliable and reproducible findings get achieved. The sample material is quite valuable. It is beneficial to prevent potential error sources.

Effect of cyclers' edging: Gradient and temperature variations across the plate may impact efficiency and result in nonspecific amplifications. PCR cycler and consumables might result in nonspecific amplifications, yield loss, and compromise the repeatability and reliability of results. All templates must get treated identically.

Incompatible leachables and wrapped plates: During routine laboratory use, plasticizers, biocides, and slip agents can separate plastic materials and taint results. Poor-quality plates get bents by heating and cooling cycles. It hinders the hand purification of beads and automation procedures.

Under- or over-shoot: After heating or cooling ramps, too-high (overshot) or too-low (undershoot) temperatures produced by block inertia negatively impact the PCR. Overshoot at the onset of denaturation eliminates enzyme activity and polymerase performance. One potential outcome is loss of yield, up to the inability to detect any amplification. Undershoots at the onset of annealing reduces the specificity of primer annealing and lead to the formation of nonspecific amplification products.