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 quantitative accuracy 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. 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.

Amplicon sequencing libraries for Rhesus box regions and for RHD/RHCE exon 9 regions were prepared for twelve combinations of genomic DNA mixtures of two individuals (A and B) at a 10:1 ratio, which served as approximation models of cfDNA from RhD-negative pregnant women. The libraries were subjected to paired-end sequencing (151 bp × 2) on a MiSeq system (Illumina) using MiSeq Reagent Kit v2 Nano. Fastq flies (.fastq.gz) for Read 1 and Read 2 were generated for each of the libararies using the Generate FASTQ workflow of the MiSeq Reporter v2 software (Illumina). Each of four zip files (TableS2_Rh_box.zip, TableS2_exon9.zip, TableS3_Rh_box.zip, TableS3_exon9.zip) contains twelve pairs of fastq files corresponding to the twelve combinations of genmic DNA mixtures. TableS2 and TableS3 filesets correspond to the libraries prepared by the one-step PCR whiout UMI and those prepared by the linear and PCR amplification protocol with UMI.