Comparison of WGA methods for genotyping fetal nucleated red blood cells for the application of non-invasive prenatal diagnosis Zhouwei Huang1, Angela N. Barrette1, Aniza P. Mahyuddin1, Sherry SY Ho2, Chia-Pin Chang3, Mahesh Choolani1 1 Department of Obstetrics & Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2 Molecular Diagnosis Centre, National University Hospital, Singapore 3 Department of Bio-Electronics, Institute of Microelectronics, A*STAR, Singapore INTRODUCTION RESULTS Fetal cells are rare in the maternal circulation, with reported frequencies ranging from 10-4 to 10-8 1-2. To potentially utilize these cells for the application of non-invasive prenatal diagnosis (NIPD), whole genome amplification (WGA) of the isolated fetal cells prior to the DNA analysis is usually necessary. Our group has chosen first trimester fetal nucleated red blood cell (FNRBC) as a potential target for developing NIPD. In the scenario of analyzing the potential “fetal” cells isolated from the maternal blood, a genotyping step for confirmation of fetal origin is thought to be important before further genome analysis could be carried out. This study aimed to compare the performance of three state-of-the-art WGA methods (PicoPLEX, MALBAC and Single Cell GenomiPhi) in genotyping single or multiple FNRBCs using Insertions and Deletions (INDELs). I. WGA of FNRBCs using three methods DNA fragment sizes of WGA products generated by the three kits varied, being GenomiPhi > MALBAC > PicoPLEX (Figure 1). There was no significant size difference between WGA-DNA from one, two or five FNRBCs using the same kit. II. Multiplex PCR with INDEL primers PCR products with the designed INDEL primers are ideally from 80 to 120bp. Our results show that the amplicon sizes from all tested samples fell into the expected range (Figure 2). Compared to fetal genomic DNA, WGA-DNA generated by PicoPLEX and by MALBAC had more similar DNA banding pattern than that generated by GenomiPhi. III. INDEL genotyping of WGA-DNA samples Figure 1. WGA-DNA of 1, 2 or 5 FNRBCs amplified using PicoPLEX, MALBAC and GenomiPhi respectively. Figure 2. Multiplex PCR products from WGA-DNA amplified using PicoPLEX, MALBAC and GenomiPhi respectively. FG = fetal genomic DNA METHODS i. Sample collection FNRBCs were extracted from first trimester placental tissues obtained post termination of pregnancy with patient consent. Peripheral blood from the same patient was also collected for maternal genomic DNA extraction. Fetal genomic DNA was extracted from trophoblasts obtained from placental tissues. Single or multiple FNRBCs were collected in PCR tubes using a micromanipulator, and immediately frozen at -80oC until further processing. ii. Whole genome amplification WGA of single, two or five FNRBCs extracted from four biological samples were performed respectively using three commercial kits, namely PicoPLEX WGA kit (Rubicon Genomics), MALBAC WGA kit (Yikon Genomics) and Single Cell GenomiPhi DNA amplification kit (GE Life Sciences). DNA quantitation was done with NanoDrop. Table 1 shows some general facts of each WGA method. Table 1. General facts of the three WGA methods iii. Genotyping with INDELs Whole genome amplified DNA (WGA-DNA) and genomic DNA samples underwent multiplex PCR reactions with primers specific to 43 INDELs and ZFX/ZFY. PCR amplicons were sequenced using next generation sequencing on Illumina Miseq platform. INDEL genotype of maternal genomic DNA, fetal genomic DNA and WGA-DNA samples were determined respectively. By matching the WGA-DNA INDEL genotype profile to the respective maternal and fetal genomic DNA profiles, the efficiency (number of INDELs amplified) and accuracy (number of INDELs correctly called) of INDEL genotyping were evaluated across the three WGA methods as well as different starting numbers of cells for the WGA. Overall, WGA-DNA generated by PicoPLEX resulted in the highest number of successfully amplified INDEL sites, as well as the highest number of correctly called INDEL genotype after matching to the INDEL profile of fetal genomic DNA and maternal genomic DNA (Table 2, Figure 3). When comparing the WGA-DNA from single cell, two cells or five cells amplified using the same kit, the five cell group consistently gave a better genotyping results compared to the lower cell number groups. INDELs Amplified INDELs with Correct Genotype Differential INDELs Amplified Differential INDELs with Fetal Genotype ZFX/ZFY Maternal gDNA 43 16 XX Fetal gDNA PicoPLEX-1C 20 10 5 3 PicoPLEX-2C 12 8 No amp PicoPLEX-5C 28 11 7 MALBAC-1C 2 MALBAC-2C 4 MALBAC-5C 19 6 GenomiPhi-1C GenomiPhi-2C 9 GenomiPhi-5C 29 17 Table 2. INDEL genotyping results from a representative female sample. Differential INDELs: INDELs that have different genotype between the mother and fetus. No amp = no amplification Kit Name WGA Principle Protocol Length Product Size (bp) PicoPLEX PCR-based amplification 3 hours, 3 steps 100 – 1000 MALBAC Multiple annealing and looping based amplification cycles, PCR-based 4 hours, 3 steps 300 - 2000 Single Cell GenomiPhi Isothermal amplification with ɸ29 DNA polymerase, non-PCR based 2.5 hours, 2 steps Average > 10K Figure 3. Comparison of INDEL genotyping among the three kits (PicoPLEX, MALBAC and GenomiPhi) and different cell number groups (1, 2 and 5 cells). CONCLUSIONS Our data suggest that for genotyping first trimester FNRBCs using INDELs, PicoPLEX may be a more suitable kit than the other two. The starting number of FNRBCs used for WGA, especially for very small numbers, would affect the efficiency and accuracy of downstream DNA analysis. WGA-DNA from as few as five cells had much improved genotyping results than from single cells. For the application of NIPD, a larger panel of INDELs may be required for genotyping purpose. References 1. Price JO, Elias S, Wachtel SS et al. (1991) Am J Obstet Gynecol 165: 1731-1737. 2. Hamada H, Arinami T, Kubo T et al. (1993) Hum Genet 91: 427-432. Contact Information Huang Zhouwei: obghz@nus.edu.sg Mahesh Choolani: mahesh_a_choolani@nuhs.edu.sg