Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing

Area of Science:

• Genomics and Molecular Biology
• Bioinformatics and Computational Biology

Background:

• Next-generation sequencing (NGS) library preparation involves attaching short oligonucleotides (adaptors) to RNA and DNA samples.
• Existing adaptors can incorporate functional elements like sample indexes and unique molecular identifiers to aid library analysis.

Purpose of the Study:

• To introduce Control Library Adaptors (CAPTORs) designed to measure the accuracy and reliability of next-generation sequencing (NGS).
• To demonstrate the utility of CAPTORs in enhancing various NGS applications, including nanopore sequencing and clinical diagnostics.

Main Methods:

• CAPTORs are integrated into the library preparation workflow for both RNA and DNA samples.
• Encoded information within CAPTORs is retrieved during the sequencing process.
• CAPTORs are applied to assess nanopore sequencing accuracy, evaluate metagenomic and RNA sequencing performance, and improve inter-sample normalization.

Main Results:

• CAPTORs effectively measure the accuracy of nanopore sequencing.
• Quantitative performance of metagenomic and RNA sequencing is evaluated using CAPTORs.
• CAPTORs improve normalization between samples and demonstrate potential for clinical applications, including correcting sequencing errors for BRCA1/2 variant detection in breast cancer.

Conclusions:

• CAPTORs provide a simple and effective method to increase the accuracy and reliability of NGS.
• This approach enables robust comparisons across different samples, reagents, and laboratories.
• CAPTORs support the expanded use of nanopore sequencing for clinical diagnostic purposes.