Abstract
Measurement of cell-free fetal DNA (cffDNA) is an indispensable process for non-invasive prenatal screening (NIPS). According to recent studies, cffDNA in maternal plasma can be enriched for various lengths of fragments, and a sufficient amount of cffDNA can effectively eliminate background interference on the part of maternal DNA. Therefore, we developed a simple and effective separation method, improved NIPS (iNIPS), that enriches the fetal fraction and improves the accuracy of NIPS for fetal aneuploid detection. We adopted a novel strategy to achieve enrichment of 125–135 bp cell-free DNA (cfDNA) by e-gel electrophoresis. To evaluate clinical performance, we compared NIPS and iNIPS results from 2153 retrospective clinical samples. Of the 22 samples with NIPS results of “no call”, 17 samples were reclassified as “unaffected” (9 cases of chr13, 5 cases of chr18, and 3 cases of chr21); 2 samples remained classified as “no call” (1 case of chr18 and 1 case of chr21); and 3 samples were identified as T21 by iNIPS. The average increase in abundance of cfDNA fragments of 125–135 bp was 2.5 times, and the average decrease in maternal background interference was 1.3 times. On this basis, the detection of fetal aneuploidy was highly improved with the fetal fraction as low as 2%; iNIPS achieved 100% sensitivity and 99.90% specificity in retrospective samples.
Highlights
The discovery of cell-free fetal DNA in maternal plasma has greatly promoted the development of non-invasive prenatal screening (NIPS) applications[1], including chromosomal microdeletion detection, microduplication detection[2,3,4,5], aneuploidy detection[6,7,8,9] and monogenic disease[2,10,11,12,13]
Recent studies have shown that when lengths of cell-free fetal DNA (cffDNA) fragments are less than 300 bp, approximately 50% of the cffDNA fragments are located within the range of 100–300 bp, and approximately 20% of maternal cellfree DNA (cfDNA) fragments are greater than 300 bp[16,17,18]
The significant difference between fetal DNA and maternal DNA in cfDNA is that the fetal DNA has a reduced peak at 166 bp and an enhanced peak at less than 150 bp[2,12,21]
Summary
The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has greatly promoted the development of non-invasive prenatal screening (NIPS) applications[1], including chromosomal microdeletion detection, microduplication detection[2,3,4,5], aneuploidy detection[6,7,8,9] and monogenic disease[2,10,11,12,13]. CffDNA is mixed with maternal-derived cfDNA that produces significant background interference. These limitations restrict the application of cffDNA. Current NGS methods for NIPS require that the proportion of cffDNA fragments in the total free plasma DNA fragments of pregnant women be greater than 4%13. Instead of removing large DNA fragments greater than 300 bp, we developed a novel strategy to improve the relative abundance of fetal-derived cfDNA by using e-gel electrophoresis to select fragments with a range less than 150 bp. Our aim was to enrich the fraction of fetal DNA in the sequencing library and reduce false-negative and false-positive rates without increasing the cost of detection
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