High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Short-read sequencing technologies face limitations in accurately sequencing large genomic regions.
• High-throughput sequencing of complex genomic targets like ribosomal RNA operons is crucial for microbial community analysis.

Purpose of the Study:

• To develop and validate a high-throughput amplicon sequencing approach for accurate, long-read sequencing.
• To combine unique molecular identifiers (UMIs) with long-read sequencing platforms (ONT and PacBio CCS) for enhanced accuracy.

Main Methods:

• Implemented a novel amplicon sequencing strategy integrating UMIs with Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) circular consensus sequencing (CCS).
• Applied the method to sequence large genomic regions, including ribosomal RNA operon amplicons (~4,500 bp) and longer genomic DNA sequences (>10,000 bp).
• Analyzed sequencing data to determine chimera rates and UMI consensus error rates at various read coverages.

Main Results:

• Achieved high-accuracy single-molecule consensus sequences for large genomic regions with a chimera rate below 0.02%.
• Demonstrated that a mean UMI consensus error rate below 0.01% can be reached with specific read coverages: 15× for ONT R10.3, 25× for ONT R9.4.1, and 3× for PacBio CCS.
• Reported mean error rates of 0.0042% (ONT R10.3), 0.0041% (ONT R9.4.1), and 0.0007% (PacBio CCS).

Conclusions:

• The developed UMI-based high-throughput sequencing approach effectively overcomes limitations of short-read technologies for large genomic regions.
• This method provides highly accurate consensus sequences, significantly reducing errors and chimeras in amplicon sequencing.
• The findings offer a robust solution for deep sequencing of large genomic targets in microbial genomics and other fields.