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Next-generation Sequencing

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....

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Updated: Dec 10, 2025

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Published on: June 21, 2018

RH genotyping by nonspecific quantitative next-generation sequencing.

Marianne Stef¹, Katie Fennell¹, Izaskun Apraiz²

¹Grifols Diagnostic Solutions Laboratories, San Marcos, Texas, USA.

|September 2, 2020

Summary

This summary is machine-generated.

Nonspecific sequencing using RH-consensus primers accurately detects and quantifies RH variants, including complex genotypes and copy number variations, with high sensitivity and specificity.

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Area of Science:

Genetics
Molecular Biology
Bioinformatics

Background:

Conventional sequencing methods for RH variants offer qualitative zygosity assessment.
Whole-genome and whole-exome sequencing provide quantitative zygosity but are more complex.
Nonspecific sequencing offers a novel approach using RH-consensus primers for variant detection and copy number quantification.

Purpose of the Study:

To evaluate a novel nonspecific sequencing method utilizing RH-consensus primers.
To assess the method's accuracy in detecting and quantifying RH variants.
To determine its capability in identifying complex RH genotypes and copy number variations.

Main Methods:

Analysis of 278 diverse genotyped samples using next-generation sequencing with RH-consensus primers.
Development of custom software for variant detection and RH genotype inference.
Evaluation of quantitative accuracy, genotype discrimination, variant detection accuracy, and RHD/RHCE assignment.

Main Results:

Observed ratio medians deviated by 3% or less from expected ratios, indicating balanced amplification.
Discriminatory power showed averages separated by 4+ standard deviations.
Variant detection sensitivity and specificity exceeded 99%; RHD/RHCE assignment was >72% sensitive and >99% specific.
Successful identification of complex genotypes and copy number variations.

Conclusions:

Nonspecific sequencing integrates genetic variation detection and quantification into a single assay.
The method demonstrates reliable quantitative performance, high sensitivity, and specificity.
It is effective for identifying complex RH genotypes.