The performance of homopolymer detection using dichromatic and tetrachromatic fluorogenic next-generation sequencing...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Homopolymer (HP) regions are challenging for next-generation sequencing (NGS) due to high error rates, including insertions, deletions, and substitutions.
• Evaluating the performance of different NGS platforms in accurately sequencing these HP regions is crucial for reliable genomic analysis.

Purpose of the Study:

• To assess the performance of dichromatic and tetrachromatic fluorogenic NGS platforms in sequencing homopolymeric regions.
• To compare the accuracy of MGISEQ-2000, MGISEQ-200, and NextSeq 2000 platforms in HP sequencing.
• To investigate the impact of unique molecular identifiers (UMIs) on improving HP sequencing accuracy.

Main Methods:

• Construction of a plasmid containing homopolymeric regions.
• Serial dilution of the plasmid to create samples with varying HP frequencies (3%, 10%, 30%, 60%).
• Sequencing of these samples on MGISEQ-2000, MGISEQ-200, and NextSeq 2000 platforms, with and without a unique molecular identifier (UMI) pipeline.

Main Results:

• A negative correlation was observed between HP length and detected frequencies across all tested platforms.
• All 8-mer HPs showed significantly decreased detection rates in all systems, except for NextSeq 2000 at 3% frequency.
• The application of UMIs largely normalized detected frequencies to expected values, with minor exceptions for poly-G on the MGI 200 platform, indicating improved performance.

Conclusions:

• The MGISEQ-2000 and NextSeq 2000 platforms demonstrated comparable performance in sequencing homopolymeric regions.
• Introducing unique molecular identifiers (UMIs) is a highly effective strategy for enhancing the accuracy of NGS platforms in sequencing homopolymeric regions.
• This study provides valuable insights into optimizing NGS workflows for regions prone to sequencing errors.