Dennis Lo's team at the Chinese University of Hong Kong, which pioneered a shotgun sequencing approach to noninvasively detect fetal aneuploidies from maternal plasma, has tested a new targeted sequencing approach to detect trisomy but has found it inferior to the previous method.
While a targeted approach could have the advantage of being higher throughput and less costly than shotgun sequencing, Lo told Clinical Sequencing News that for now, he plans to stick with the shotgun approach because the targeted approach is "less robust."
Currently, Sequenom, LifeCodexx, and Verinata Health all have tests on the market that use the shotgun sequencing approach, while Ariosa Diagnostics offers a prenatal trisomy test based on a targeted sequencing approach. However, Ariosa CEO Ken Song told Clinical Sequencing News that the company's method differs substantially from the method described in Lo's paper, and Ariosa has demonstrated that its test is highly sensitive and specific.
Natera has also said it is developing a targeted sequencing-based approach for prenatal trisomy detection, but has not discussed the details of its strategy.
The main difference between the shotgun sequencing strategy and Lo's targeted sequencing strategy is that the latter method analyzes SNPs in order to detect trisomy, while the shotgun sequencing approach counts chromosomes.
In Lo's recent study, which was published this week in PLoS One, the researchers used Agilent SureSelect to enrich for 2,906 SNP loci on chromosomes 7, 13, 18, and 21 and then sequenced the samples to 225-fold coverage on the Illumina Genome Analyzer.
The team screened 14 pregnant women, seven of whom were positive for trisomy 21, and genotyped the maternal and fetal samples.
Following sequencing, the team then examined the different SNP loci, first separating out the informative SNPs — those that were homozygous in the mother and heterozygous in the fetus as determined by genotyping. From these SNPs, the researchers determined fetus-specific alleles.
The informative SNPs were then used to calculate the ratio between fetus-specific alleles and shared alleles for chromosome 21 to the ratio between the fetus-specific alleles and shared alleles for the reference chromosomes, which included chromosomes 7, 13, and 18.
A ratio of 1 indicates euploid. If a fetus is positive for trisomy 21, and the extra chromosome is contributed by the father, the ratio should be 2, while maternally derived trisomy 21 should yield a ratio of less than 1.
Of the seven affected fetuses, the method was able to accurately call the one paternally derived trisomy case, but could not distinguish the maternally derived trisomy cases. Furthermore, when using a computer model to evaluate 1,000 cases, the model yielded a mean fetal-specific ratio for paternally derived trisomy of 2.35, which could be distinguished from the reference of 0.91. However, the mean fetal-specific ratio for maternally derived trisomy 21 was 1.10, which overlapped with the euploid cases.
Lo's team then demonstrated that detection could be improved by increasing the sequencing depth, as well as the number of SNP loci that are sequenced, which would increase the number of informative SNPs. The researchers determined that in order to achieve sensitivity and specificity greater than 99 percent, they would require 130,000 informative SNPs in the case of maternally derived trisomy 21 and 1,100 informative SNPs in paternally derived trisomy 21.
Lo said that the fact that not all the targeted SNPs are informative is a main weakness of the method. "A large proportion of the sequenced reads would be wasted," he said, compared to the shotgun sequencing method in which "virtually all uniquely mappable reads can be used."
Another disadvantage of this targeted approach is that it requires parental genotype information, while the shotgun sequencing strategy does not.
While Ariosa's recently launched Harmony Prenatal Test uses a targeted sequencing strategy to diagnose fetal trisomy 21, CEO Song told Clinical Sequencing News that the method described by Lo is "completely different than the approach we take."
The main difference, he said, is that Ariosa's method does not rely solely on looking at polymorphic differences. Ariosa uses polymorphic differences only to estimate the fraction of fetal DNA that is present in the maternal plasma.
To make trisomy calls, the company looks at the number of reads from each of the targeted chromosomes. Then, it incorporates the fraction of fetal DNA and risk information related to maternal age into an algorithm to come up with a patient-specific odds ratio of trisomy versus disomy.
Ariosa has described its internally developed assay — digital analysis of selected regions, or DANSR — most recently in the American Journal of Obstetrics and Gynecology, demonstrating that from 400 pregnancies it accurately called all 50 trisomy 21 cases, as well as 49 out of 50 trisomy 18 cases with no false positives (CSN 2/1/2012).
Song did agree that the targeted approach was "not as straightforward as a shotgun sequencing approach," noting that "Anyone that wants to develop a targeted approach will face challenges to make it robust."