54. IMPLEMENTATION OF FIRST NIPT SERVICE LABORATORY IN AN INDIAN HOSPITAL

Abstract

Introduction Non-Invasive Prenatal Testing (NIPT) for common fetal aneuploidies is rapidly increasing in India for both naturally conceived pregnancies and pregnancies conceived by assisted reproductive technologies (ART). This study aims to establish the very first next generation sequencing (NGS) laboratory for NIPT in an Indian hospital using the Ion Torrent platform to provide a complete in-house solution for pregnant women. Materials and Methods In brief, all plasma DNA samples (both control and test groups) were converted to genomic DNA libraries using Fragment Library Kit (Thermo Fisher Scientific, Cat. No. 4471252) with Ion Xpress Barcode-Adapters mix (Thermo Fisher Scientific, Cat. No. 4471250) using our laboratory developed protocol. The libraries were then quantified using Qubit dsDNA HS (high sensitivity) Assay Kit, (Thermo Fisher Scientific) and diluted to equimolar amounts before pooling. The Ion Chef System (Thermo Fisher Scientific), as part of the workflow, was used for automated template preparation and loading the sequencing chip. Thereafter, low-pass whole-genome sequencing was done using the ion semiconductor sequencer Ion S5 System (Thermo Fisher Scientific,) to achieve at least 5 million sequence reads. In the dry-lab, the sequenced reads were aligned to the reference human genome (UCSC hg19) locally and their BAM files were generated. The euploid BAM files from the training set of samples were assigned to NIPT reference group and their aligned reads were partitioned into 50,000 bp bins. The number of reads in each bin becomes the unit for comparison with the test samples. GC corrections were performed based on the average coverage of genomic regions having a similar GC-content to correct the coverage differences between bins having a different GC percentage. In this validation study, the standard Z-score prediction models returned a value between -3 and +3 for all the chromosomes, indicating that there is no trisomy present. The scores for aneuploidy samples all showed a value above 4. The z-score refers to the number of standard deviations from the mean of a reference data set and in all molecular counting approaches, a z-score of more than 4 is considered a positive result. Results The algorithm was blindly tested on a cohort of 100 known samples, of which the 12 were T21, eight were T18, four were T13 and 76 were from euploid pregnancies. Our algorithm correctly called all euploid and aneuploid cases. For all aneuploidies, z scores were above a value of 4. Interestingly, accurate detection of aneuploidy was also noted at foetal fraction as low as 3.2%. Therefore, based on our findings, our algorithm is expected to be sensitive and specific for up to 99.9% of pregnancies with one of the three common trisomies. DISCUSSION Based on the high performance of our NIPT test in validation studies, testing has commenced at Lifeline Hospital, including pregnancies conceived by ART. Clinical performance is currently under evaluation to derive positive predictive value (PPV) and negative predictive value (NPV) for each trisomy.