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

ABSTRACT

OBJECTIVES: Homopolymer (HP) sequencing is error-prone in next-generation sequencing (NGS) assays, and may induce false insertion/deletions and substitutions. This study aimed to evaluate the performance of dichromatic and tetrachromatic fluorogenic NGS platforms when sequencing homopolymeric regions. RESULTS: A HP-containing plasmid was constructed and diluted to serial frequencies (3%, 10%, 30%, 60%) to determine the performance of an MGISEQ-2000, MGISEQ-200, and NextSeq 2000 in HP sequencing. An evident negative correlation was observed between the detected frequencies of four nucleotide HPs and the HP length. Significantly decreased rates (P < 0.01) were found in all 8-mer HPs in all three NGS systems at all four expected frequencies, except in the NextSeq 2000 at 3%. With the application of a unique molecular identifier (UMI) pipeline, there were no differences between the detected frequencies of any HPs and the expected frequencies, except for poly-G 8-mers using the MGI 200 platform. UMIs improved the performance of all three NGS platforms in HP sequencing. CONCLUSIONS: We first constructed an HP-containing plasmid based on an EGFR gene backbone to evaluate the performance of NGS platforms when sequencing homopolymeric regions. A highly comparable performance was observed between the MGISEQ-2000 and NextSeq 2000, and introducing UMIs is a promising approach to improve the performance of NGS platforms in sequencing homopolymeric regions.