Application of NGS technologies to Preimplantation Genetic Diagnosis (PGD)

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  • Slide7. Let’s talk about using Next-Generation Sequencing in PGD. Like before, biopsy, cell lysis and whole genome amplification. In this case, we can take an aliquot of the amplified genome for the detection of monogenic diseases and amplify the gene or region of interest by PCR. So, here it is possible to screen for aneuploidy and detect specific mutations that lead to monogenic diseases at the same time! /// Afterwards, DNA is fragmented and an adapter is added.

    Slide8. Sequencing is multiplexed. In other words, the DNA fragments of each embryo to analyze, besides of the adapter, have a barcode sequence that identifies the sample. It allows sequencing of different embryos at the same chip.

    Slide9. After sequencing, it is necessary to make a GC bias correction. As you know, the number of reads depends on the GC content of the region, and there are chromosomes with more GC content than the others (you can see it in this bar plot). This data has been obtained using seqtk and the awk script below the plot along the human genome version 37. Afterwards, reads are aligned (aligners: The Torrent Mapping Alignment Program for Ion Torrent (TMAP) or BWA are examples).

    Slide10. Data is visualized using the local genome browser IGV. In this picture we can see the coverage across the different chromosomes. The coverage is analyzed by a statistical model based on a Hidden Markov Model where the states are the ploidy status (the number of chromosomes). There is monosomy of chromosomes 5 and 22 shown by this picture and the embryo is a female.

    Slide11. As the IGV genome browser is an open-source application, IGV has been customized for karyotype view.

    Slide12. With Ion Torrent, it has been proven that only 150K reads per embryo are necessary in order to detect aneuploidy, with a coverage average of 0.01X. This coverage is enough, because we are looking at big genomic variations such as aneuploidy instead of calling SNPs that requires a lot more coverage. We see as well perfect concordance between aCGH results and results based on sequencing. Low coverage shorten the workflow timing to 15 hours and, together with the possibility to sequence 32 embryos at a time per run, makes possible to decrease the price of PGD down to 70€/embryo. It is a much lower price than PGD based on microarray.

    Slide13. Moreover, PGD based on NGS allows at the same time the detection of aneuploidy and mutations of monogenic diseases. The latter case, if you remember from some slides before, requires an additional PCR step after WGA in order to have high coverage of the region or gene of interest. So for example, at the picture of your right, there are a normal embryo, an embryo carrier for the indel Δl507 and an embryo carrier of the SNP D1152H. Both are cause of cystic fibrosis.

    Slide14. Prenatal diagnosis here. It is not a PGD because the women is already pregnant, but it allows to detect aneuploidy by simply doing a blood test. Cell-free DNA from the fetus has been found in the plasma of pregnant women. So, cell-free DNA from the sample is sequenced, aligned to the genome, a GC bias correction is performed and finally, a Z-test is carried out. /// The basis is that a maternal plasma sample from a pregnancy with a trisomy 18 is expected to have higher genomic representation of chromosome 18 compared with the references, which are women pregnant with euploid fetuses. In the Z-test, the observation is the genomic representation of the chromosome of interest in the test sample, while the mean and SD refers to the genomic representation of the chromosome of interest in the references. A Z-score above 3 is considered aneuploidy.
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  • Slide2. Back to the 1970s in the United Kingdom, two parents (Lesley and John Brown) tried for 9 years to get pregnant. After this time, Lesley underwent an experimental procedure called In-Vitro Fertilization (IVF) that succeed. The parents had a daughter, Louise Brown, born in 1978. She was the first baby born using IVF.

    Slide3. In the middle of last year 2013, Connor Levy was born. His parents, Marybeth and David signed up for IVF in Pennsylvania. They underwent IVF such as the case of Louise brown, but in this case the embryos were tested for abnormal number of chromosomes using Next Generation Sequencing. This process is called Preimplantation Genetic Diagnosis (PGD).

    Slide4. First of all, let’s talk about IVF. It consists in ovarian stimulation and aspiration of oocytes. Then these oocytes are fertilized by sperm and the zygotes are cultured for 3-6 days. After this time, 1-2 of the best looking embryos under microscope are transferred to the mother’s uterus.

    Slide5. However, the success rate of IVF is low specially for women above 35 years old. For this subgroup of patients, there are low implantation rates and high pregnancy loss. It has been seen that aneuploidy in embryos increases with maternal age, and over 80-90% of early pregnancy losses the embryos had an abnormal number of chromosomes. So, it is known that the older is the women, the higher rate of aneuploidy and, therefore, higher rate of miscarriages. The use of PGD in IVF cycles has increased implantation rates and decreased the number of pregnancy losses.

    Slide6. So, if a PGD is performed during the IVF process, a biopsy of the embryo must be performed in order to extract one cell. The PGD needs to be fast, because the embryo is under incubation and needs to be implanted. After biopsy, cell lysis followed by Whole Genome Amplification (WGA) is performed. Then, DNA is fluorescently marked and ready to be hybridized to the microarray versus a control sample. The most common method to perform a PGD today is a microarray-based Comparative Genomic hybridization (aCGH). After hybridization, the microarray is scanned and we obtain the results: at the picture we see a trisomy at chromosome 10, and the embryo is a female compared to a male control. The workflow is between 16 and 24 hours and the price quite expensive. /// Sometimes PGD is performed not to screen for copy-number abnormalities, but to detect monogenic diseases (mutations in one gene) that one or both parents have with the aim of having a healthy child. In this case, we cannot screen for aneuploidy at the same time due to we have tiny amounts of DNA from one cell. /// The question is: could we use a method other than aCGH in order to decrease timing and price?
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Application of NGS technologies to Preimplantation Genetic Diagnosis (PGD)

  1. 1. Application of NGS technologies to Preimplantation Genetic Diagnosis (PGD) Andreu Paytuví MSc in Bioinformatics, UAB
  2. 2. Little bits of history 2/12
  3. 3. Little bits of history First baby born from NGS! 3/12
  4. 4. Why PGD? Pictures from: Fertility Institute of New Jersey & New York. http://www.center4ivf.com/ (Accessed 28/02/2014). 4/12
  5. 5. Why PGD? Pictures from: Chromosome Screening. http://www.chromosome-screening.org/ (Accessed 28/02/2014). 4/12 Exampleofaneuploidy: Turnersyndrome
  6. 6. Why PGD? Pictures from: Theisen, A. (2008) Microarray-based comparative genomic hybridization (aCGH). Nature Education 1(1):45 aCGH 2500-3500€ for 6-10 embryos ≈350-400€/embryo Workflow: 16-24h 10 X Y 5/12
  7. 7. PGD based on NGS 6/12
  8. 8. PGD based on NGS Sequencing 7/12
  9. 9. PGD based on NGS 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Percentage(%) Chromosomes seqtk comp hs37m.fa.gz | awk '/^[0-9MXY]/{x=$4+$5;y=x+$3+$6;print $1"t"x/y}' Sequencing GC bias correction 7/12
  10. 10. Data analysis Picture from: Rico, Alan. Pre-implantation Genetic Testing on Ion Torrent™ PGM™ System. Ion Tour 2013, Life Technologies. Microsoft PowerPoint file. Based on Hidden Markov Model (HMM): - States: Ploidy status (monosomy, disomy, etc.). - Observations: coverage (1X, 2X, 5X, 10X, etc.). 8/12
  11. 11. Data analysis Picture from: Rico, Alan. Pre-implantation Genetic Testing on Ion Torrent™ PGM™ System. Ion Tour 2013, Life Technologies. Microsoft PowerPoint file. Based on Hidden Markov Model (HMM): - States: Ploidy status (monosomy, disomy, etc.). - Observations: coverage (1X, 2X, 5X, 10X, etc.). 8/12
  12. 12. Data analysis Ion PGM™ 150K reads aCGH Up to 32 embryos per run ≈70€/embryo Workflow: ≈15h Pictures from: Rico, Alan. Pre-implantation Genetic Testing on Ion Torrent™ PGM™ System. Ion Tour 2013, Life Technologies. Microsoft PowerPoint file. 9/12
  13. 13. Data analysis CFTR case Picture from: Treff, N. 2013. Evaluation of targeted next-generation sequencing–based preimplantation genetic diagnosis of monogenic disease. Fertility And Sterility, 99:5, p. 1377-1390. Picture from: Rico, Alan. Pre-implantation Genetic Testing on Ion Torrent™ PGM™ System. Ion Tour 2013, Life Technologies. Microsoft PowerPoint file. 10/12
  14. 14. Prenatal diagnosis Pictures from: Bianchi, D. W. 2012. From prenatal genomic diagnosis to fetal personalized medicine: progress and challenges. Nat Med., 18, 1041–1051. Alkan, C., et al. 2010. Personalized Copy-Number and Segmental Duplication Maps using Next-Generation Sequencing. Nat Genet., 41(10), 1061–1067. Z score of an observation is the number of standard deviations it falls above or below the mean. -Z score of mean = 0 -Cut-off value of 3 (|Z| > 3) LOESS method (GC correction) x --> region y’ --> corrected counts f(x) --> mean of counts for that GC% e(x) --> expected counts (overall average) 11/12
  15. 15. Other references: Recommended talks: Munne, S., et al. 2005. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertility and Sterility, 84(2), 331-335. Handyside, A. H. 2013. 24-chromosome copy number analysis: a comparison of available technologies. Fertility and Sterility, 100(3), 595–602. Life Technologies, Aneuploidy detection by low-pass whole-genome sequencing on the ion PGM™ system, Appl. Note CO06456 0713. Chen, E. Z., et al. 2011. Noninvasive Prenatal Diagnosis of Fetal Trisomy 18 and Trisomy 13 by Maternal Plasma DNA Sequencing. PLoS One, 6(7), e21791. Thanks for your attention!

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