1. © Copyright 2009 by the American Association for Clinical Chemistry Maternal Plasma DNA Analysis Using Massively Parallel Sequencing-By- Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21 Rossa W.K. Chiu, Hao Sun, Ranjit Akolekar, Christopher Clouser, Clarence Lee, Kevin Mckernan, Daixing Zhou, Kypros H. Nicolaides, and Y. M. Dennis Lo March 2010 http://www.clinchem.org/cgi/content/full/56/3/460 © Copyright 2010 by the American Association for Clinical Chemistry Journal Club

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Prenatal Diagnosis A part of obstetrics care Fetal tissue for genetic analysis is obtained conventionally by amniocentesis or chorionic villus sampling The procedures are invasive with risk of fetal miscarriage

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Fetal DNA in Maternal Plasma First reported in 1997 Exists as short fragmented molecules in cell-free form Only amounts to ≈ 10% of the total DNA among a background of maternal DNA Successfully used for noninvasive prenatal assessment of fetal sex and fetal rhesus D status

4. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Trisomy 21 Typically due to presence of a third copy of chromosome (chr) 21 in fetal genome Incidence ≈ 1 out of 800 pregnancies Other aneuploidies: trisomy 18, trisomy 13, Turner syndrome

5. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why is it more challenging to achieve noninvasive prenatal diagnosis of fetal trisomy 21 by fetal DNA analysis in maternal plasma compared with applications such as fetal sex determination?

6. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Massively Parallel Genomic Sequencing Plasma was obtained from pregnant women DNA was extracted Universal adaptors were added to the ends of the plasma DNA molecules Clonal amplification by emulsion PCR Random sequencing of DNA using sequencing-by-ligation 50 bp from one end of each plasma DNA molecule sequenced

7. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  Bioinformatics Analysis 35 bp of each sequenced read was mapped to the repeat- masked reference human genome Only reads that perfectly mapped to one location of the reference genome were included for further analysis Chromosomal origin of each included read was recorded

8. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  Chromosomal Genomic Representation (GR) GR of chrN = %chrN = # reads from chrN / # total reads Expected GR of chrN = nucleotide content of chrN as a proportion of the genomic content of a normal human genome  Deviation from Expected GR (experimentally derived GR for chrN) – (expected GR for chrN) expected GR for chrN  CV of GR SD of GR for chrN / mean GR for chrN

9. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  Diagnosis of Trisomy 21 >chr21 z scores %chr21 test case – %chr21 reference controls SD reference controls z score > 3 used as diagnostic cutoff to identify trisomy 21

10. © Copyright 2009 by the American Association for Clinical Chemistry Question  What factors may cause a deviation in the measured GR from the expected GR for any particular chromosome?

11. © Copyright 2009 by the American Association for Clinical Chemistry Figure 1. z scores for chromosome 21 in the control (cases 1–4, in black), euploid (cases 5–10, in blue), and trisomy 21 (cases 11–15, in red) pregnancies. A z score of 3 (dashed line) was used as a cutoff to determine the presence of overrepresentation of sequences from chromosome 21. Results

12. © Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Bar chart showing the median degree of deviation in genomic representation for the human chromosomes ordered from left to right in increasing GC contents. Results

13. © Copyright 2009 by the American Association for Clinical Chemistry Figure 3. CV for measuring the %GR of each chromosome ordered from left to right in increasing GC contents. Results

14. © Copyright 2009 by the American Association for Clinical Chemistry Question  What factors may affect the z score values and its ability in discriminating trisomy 21 from euploid cases?

15. © Copyright 2009 by the American Association for Clinical Chemistry Discussion  Noninvasive Prenatal Diagnosis of Trisomy 21 Accurate diagnosis can be achieved by massively parallel sequencing of maternal plasma DNA Pending validation by large-scale studies Pending reduction in sequencing costs and improvements in throughput

16. © Copyright 2009 by the American Association for Clinical Chemistry Discussion  Application to Trisomy 18 and Trisomy 13 Need to improve the precision for measuring GR of chromosomes 18 and 13