Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles

• Main Authors: Asuka Hori, Hiroko Ogata-Kawata, Aiko Sasaki, Ken Takahashi, Kosuke Taniguchi, Ohsuke Migita, Akihiro Kawashima, Aikou Okamoto, Akihiko Sekizawa, Haruhiko Sago, Fumio Takada, Kazuhiko Nakabayashi, Kenichiro Hata
• Format: Article
• Language: English
• Published: BMC 2021-09-01
• Series: BMC Research Notes
• Subjects: RHD; Cell-free DNA (cfDNA); Non-invasive prenatal testing (NIPT); Amplicon sequencing; Unique molecular identifier (UMI)
• Online Access: https://doi.org/10.1186/s13104-021-05793-4

Abstract Objective We aimed to simplify our fetal RHD genotyping protocol by changing the method to attach Illumina’s sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its reliability and robustness by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. Results Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal RHD genotyping for East Asian populations into clinical practice.