Next Generation Sequencing Details

Next Generation Sequencing

Next Generation Sequencing (NGS) technology can allow unprecedented, gigabase (gb)-length sequencing of genomic DNA per run. In addition to the utility of NGS in gene target resequencing and deciphering genetic biomarkers in disease, NGS-based applications (such as transcriptomics/RNA-sequencing, ChIP-sequencing, single-cell sequencing) provide a high-resolution, multidimensional approach to linking nucleic acid sequences to cell phenotypes.

Advancements in NGS sequencing chemistries and instrument throughput provides decreased cost-per-base sequencing. However, sample preparation for sequencing remains a challenge due to intractable sample types, input templates, varying quality of library preparation and cost per library. Furthermore, multiple, time-consuming steps leading to library preparation can cause bottlenecks in the sequencing process, exacerbated by insufficient DNA yield, excessive sequence bias, and other challenges to library quality that compromise sequencing data. Geneture's nucleic acid extraction kits provide a fast, cost-effective solution for the highest quality NGS sample preparation. 

Library Validation for Next Generation Sequencing 

Inaccurate quantification or poor normalization of next generation sequencing libraries prior to sequence amplification and detection can compromise results. Quantification and normalization are particularly important during multiplexing of samples or detecting intra-sample sequence variants. 

Understanding library quantification assays for next-generation sequencing applications

With the sequencing capacity of NGS instrumentation continuing to rise, researchers are able to pool more samples, or libraries, into a single sequencing run, greatly reducing the per-sample cost of sequencing. However, NGS library concentrations can vary widely, based on the amount and quality of nucleic acid sample inputs, as well as the target enrichment method that is used. In order to ensure that each pooled library is sequenced to the desired depth, NGS libraries must be carefully quantified and normalized so that each sample achieves the required number of reads.

Common library quantification methods include fluorometric spectroscopy and quantitative PCR (qPCR). While both methods provide relatively accurate measurement of library concentration, there are assayspecific considerations associated with these techniques.

Overview of methods

Qubit dsDNA HS assay The Qubit dsDNA HS assay utilizes a target-specific dye that emits fluorescence when bound to dsDNA. Unlike UV spectroscopy, which can overestimate sample concentration due to contaminants in a sample, the Qubit Fluorometer together with the Qubit dsDNA HS assay does not measure salts, nucleotides, or RNA that may be present. The Qubit assay is also much more sensitive than UV spectroscopy, as it is able to measure dilute DNA samples with significantly less noise.

The Qubit dsDNA HS assay utilizes two reference standards to generate a standard curve that is used to empirically determine the concentration of a sample. The assay can be used to measure samples between 10 pg/µL and 100 ng/µL of DNA. Samples with concentrations above this range have to be diluted and retested to ensure that they fall within the linear range of the reference standards. The Qubit dsDNA HS assay has good accuracy, with a coefficient of variation (CV) of <5% across a range of 0.5–200 ng/µL. The Qubit dsDNA HS assay workflow is quick and easy. The protocol utilizes a simple mix-and-read format with incubation times of only a few minutes. Briefly, 1–20 µL of sample (or 1–10 µL of reference standard) is added to the Qubit working solution. Following a 2-minute incubation, samples are read and the DNA concentrations in the samples are calculated.

Ascend Plant Genomic DNA Nucleic Acid Extraction Kit uses magnetic beads to adsorb DNA to achieve the purpose of rapid purification of plant genomic DNA. Plant Genomic DNA Nucleic Acid Extraction Kit 50T is suitable for extracting high-purity genomic DNA from 50 mg～200 mg plant samples, suitable for PCR, next-generation sequencing, gene chip and other molecular biology experiments.

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