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Fig. 1 | BMC Research Notes

Fig. 1

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

Fig. 1

Conventional and newly developed library preparation protocols for amplicon sequencing-based noninvasive fetal RHD genotyping. A Genomic organization of the RHD/RHCE locus. Open and closed boxes indicate upstream and downstream Rhesus boxes. Closed and open arrowheads indicate PCR primer pairs to amplify 105-bp intervals in Rhesus boxes and 148-bp intervals spanning the exon 9 and the intron 9 of RHD and RHCE genes, respectively. Amplicon-seq libraries without UMI can be prepared by our conventional ligation-based protocol (B, C) and the one-step multiplex PCR protocol (E, F) established in this study. Amplicon-seq libraries with UMI can be prepared by the newly established linear and PCR amplification protocol (H, I). Approximate total and hands-on time (B, E, H), diagrams for experimental procedures (C, F, I), and representative electropherograms of intermediate PCR products and/or final libraries (D, G, J) are shown for three protocols. In the panels C, F, and I, genomic DNA is shown by a black line; PCR primers that target genomic DNA sequences are shown by blue arrows; adaptor sequences are shown in dark/light green/orange; 12-base UMIs are shown in red. Index sequences to de-multiplex sequence reads after sequencing of pooled libraries are omitted for simplicity. Panels D, G, and J show representative electropherograms of Rhesus box amplicons (“box”), for RHD/RHCE exon 9 amplicons (“ex9”), and for multiplex amplification by two pairs of primers (“multiplex”). Horizontal and vertical axes of each electropherogram represent fluorescent intensity and DNA size (bp), respectively. Lower and upper marker peaks are present at the positions of 35 bp and 10,380 bp, respectively. In our conventional [4] and one-step PCR protocols (C, F), multiplex PCR using two pairs of primers gave rise to specific amplification of target genomic intervals (D, G). K A data analysis workflow showing the informatics tools used and their step-by-step functions. L Bam coverage tracks of the RHD genotyping results by the one-step PCR protocol (F) for three combinations of the mixtures of genomic DNA (combinations #1, #2 and #3 in Additional file 2: Table S2) and for two cfDNA samples (#46 and #59) from RhD-negative pregnant women (Table 1) visualized using IGV. The mapped read patterns indicated that the RHD genotypes of the mother (Mo) and the fetus (Fe) were RHD*01N.01/RHD*01N.01 and RHD*01/RHD*01N.01, respectively, in both cases. The vertical ranges of mapped read numbers were 0–25,000 for the top three panels, 0–40,000 for cfDNA#46, and 0–150,000 for cfDNA#59

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