Præimplantationsgenetisk test for aneuploidi på en halvlederbaseret næste generations sekventeringsplatform
Summary
Protokollen præsenterer de overordnede procedurer i laboratoriet, der kræves i genetisk test før implantation for aneuploidi på en halvlederbaseret næste generations sekventeringsplatform. Her præsenterer vi de detaljerede trin i helgenomforstærkning, udvælgelse af DNA-fragment, bibliotekskonstruktion, skabelonforberedelse og sekventering af arbejdsflow med repræsentative resultater.
Abstract
Næste generations sekventering har fået stigende betydning i den kliniske anvendelse i bestemmelsen af genetiske varianter. I den genetiske test før implantation har denne teknik sine unikke fordele i skalerbarhed, gennemstrømning og omkostninger. Til præimplantationsgenetisk test til aneuploidianalyse giver det halvlederbaserede næste generations sekventeringssystem (NGS), der præsenteres her, en omfattende tilgang til bestemmelse af strukturelle genetiske varianter med en minimumsopløsning på 8 Mb. Fra prøveoptagelse til den endelige rapport kræver arbejdsprocessen flere trin med tæt overholdelse af protokoller. Da forskellige kritiske trin kunne bestemme resultatet af forstærkning, bibliotekets kvalitet, dækning af læsninger og output af data, kunne beskrivende information med anden visuel demonstration end ord give flere detaljer til operationen og manipulationen, hvilket kan have stor indflydelse på resultaterne af alle kritiske trin. De metoder, der præsenteres heri, vil vise de procedurer, der er involveret i helgenomforstærkning (WGA) af biopsierede Trophectoderm (TE) celler, genomisk bibliotekskonstruktion, sequencerstyring og endelig generering af kopinummervarianters rapporter.
Introduction
Aneuploidi er abnormiteten i antallet af kromosomer ved tilstedeværelsen af et eller flere ekstra kromosomer eller fraværet af et eller flere kromosomer. Embryoner, der bærer en form for aneuploidi, såsom tab af et X-kromosom (Turner syndrom), ekstra kopier af autosomer, som trisomier af autosom 21 (Downs syndrom), 13 (Patau syndrom) og 18 (Edwards syndrom) eller ekstra kønskromosomer som 47, XXY (Klinefelter syndrom) og 47, XXX (Triple X syndrom), kan overleve til sigt med fødselsdefekter1. Aneuploidi er den primære årsag til aborter i første trimester og in vitro befrugtning (IVF)svigt 2. Det rapporteres, at aneuploidihastigheden kan variere fra 25.4% -84.5% gennem de forskellige alderslag i den naturlige cyklus og medicinsk kontrolgruppe i IVF-praksis3.
Næste generations sekventeringsteknologi bliver vildt anvendt til bestemmelse af genetisk information klinisk; det giver praktisk adgang til genomsekvens med effektivitet og høj gennemstrømning. Især næste generations sekventering revolutionerede også diagnosen af lidelser med genetiske faktorer og test for abnormitet i genomet4. Ved hjælp af halvledersekventeringsteknologi til direkte overførsel af kemiske signaler i sekventeringsbioreaktion til digitale data giver det halvlederbaserede sekvenssystem en direkte realtidsdetektion til sekvensdata i 3-7 h 5,6.
I en IVF-procedure undersøger præimplantationsgenetisk test (PGT) embryoets genetiske profil, inden den overføres til livmoderen for at forbedre IVF-resultatet og reducere risikoen for genetiske lidelser hos nyfødte 1,7. I PGT kombineret med NGS-teknikker forstærkes genetisk materiale ekstraheret fra mindre end 10 celler med helgenomforstærkningssæt eller et uafhængigt udviklet helgenom-amplifikationsreagens. Dette kræver kun et trin i forstærkningsfasen og kræver ikke forforstærkning for at opnå helgenomforstærkningsprodukter. Primere eller paneler til kopinummervariant og speciel genlokalisering er designet og anvendt i det konstruerede bibliotek.
En typisk arbejdsgang for præimplantationsgenetisk testning-aneuploidi (PGT-A) i NGS involverer serielle procedurer og kræver en intens arbejdsbyrde for laboratoriepersonale8. Nogle fejloperation forårsaget procedure roll-back kan føre til uønsket tab af både tid og ressourcer i laboratoriet. En kortfattet og klar standardoperationsprocedure (SOP) for PGS-NGS-arbejdsgangen er nyttig; Ordformatprotokoller kan dog ikke præsentere mere detaljerede oplysninger om prøvebehandling, enhedsmanipulation og instrumenters indstillinger, som kan visualiseres i en videoprotokol. I denne artikel kan en valideret arbejdsgang kombineret med en visualiseret demonstration af driftsdetaljer tilbyde mere direkte og intuitive henvisningsprotokoller i PGT-praksis på en halvledersekventeringsplatform.
Protokollen beskriver her en metode, der understøtter batching op til 16 embryobiopsier parallelt. For større partier anbefales det at bruge en kommerciel kitbaseret protokol til halvledersekventering, såsom Reproes-PGS.
Protocol
Representative Results
Discussion
Kromosomal aneuploidi af embryoner er årsagen til en stor del af graviditetstabet, uanset om det er undfanget naturligt eller in vitro befrugtning (IVF). I den kliniske praksis med IVF foreslås det, at screening af embryoaneuploidi og overførsel af euploidiembryoet kan forbedre resultatet af IVF. Fluorescens in situ hybridisering er den tidligste teknik, der er vedtaget til kønsselektion og PGT-A; Denne teknik kræver dog mere teknisk ekspertise fra laboratoriepersonale og er relativt arbejdskræven…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke Dr. Zhangyong Ming og Mr. Rongji Hou for deres råd om LIMS udvidet ansøgning. Denne undersøgelse er støttet af PLA Special Research Projects for Family Planning (17JS008, 20JSZ08), Fund of Guangxi Key Laboratory of Metabolic Diseases Research (nr. 20-065-76) og Guangzhou Citizen Health Science and Technology Research Project (201803010034).
Materials
| 0.45 μm Syringe Filter Unit | Merkmillipore | Millex-HV | |
| 1.5 mL DNA LoBind Tubes | Eppendorf | 30108051 | |
| 15 mL tubes | Greiner Bio-One | 188261 | |
| 2.0 mLDNA LoBind Tubes | Eppendorf | 30108078 | |
| 50 mL tubes | Greiner Bio-One | 227261 | |
| 5x Anstart Taq Buffer (Mg2+ Plus) | FAPON | ||
| Anstart Tap DNA Polymerase | FAPON | ||
| AMPure XP reagent (magnetic beads for dna binding) | Beckman | A63881 | https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/pcr/a63881 |
| Cell Lysis buffer | Southern Medical University | Cell lysis buffer containing 40 mM Tris (pH 8), 100 mM NaCl, 2 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% (v/v) Triton X-100, 5 mM sodium pyrophosphate, 2 mM β-glycerophosphate, 0.1% SDS | |
| ClinVar | NCBI | https://www.ncbi.nlm.nih.gov/clinvar/ | |
| DNA elution buffer | NEB | T1016L | |
| dNTP | Vazyme | P031-AA | |
| DynaMag-2 Magnet | Life Technologies | 12321D | |
| Ethyl alcohol | Guangzhou Chemical Reagent Factory Thermo Fisher Scientific | http://www.chemicalreagent.com/ | |
| Independently developed whole genome amplification reagents | Southern Medical University | The reagents consist of the following components: 1. Cell Lysis 2. Amplification Pre-mixed solution 1) Primer WGA-P2 (10 μM) 2) dNTP (10 mM) 3) 5x Anstart Taq Buffer (Mg2+ Plus) 3. Amplification Enzyme 1) Anstart Tap DNA Polymerase (5 U/μL) |
|
| Ion PI Hi-Q OT2 200 Kit | Thermo Fisher Scientific | A26434 | Kit mentioned in step 4.2.8 |
| Ion PI Hi-Q Sequencing 200 Kit | Thermo Fisher Scientific | A26433 | |
| Ion Proton System | Life Technologies | 4476610 | |
| Ion Reporter Server System | Life Technologies | 4487118 | |
| isopropanol | Guangzhou Chemical Reagent Factory | http://www.chemicalreagent.com/ | |
| Library Preparation Kit | Daan Gene Co., Ltd | 114 | https://www.daangene.com/pt/certificate.html |
| NaOH | Sigma-Aldrich | S5881-1KG | |
| Nuclease-Free Water | Life Technologies | AM9932 | |
| Oligo WGA-P2 | Sangon Biotech | 5'-ATGGTAGTCCGACTCGAGNNNN NNNNATGTGG-3' |
|
| OneTouch 2 System | Life Technologies | 4474779 | Template amplification and enrichment system |
| PCR tubes | Axygen | PCR-02D-C | |
| PicoPLEX WGA Kit | Takara Bio USA | R300671 | |
| Pipette tips | Quality Scientific Products | https://www.qsptips.com/products/standard_pipette_tips.aspx | |
| Portable Mini Centrifuge LX-300 | Qilinbeier | E0122 | |
| Qubit 3.0 Fluorometer | Life Technologies | Q33216 | Fluorometer |
| Qubit Assay Tubes | Life Technologies | Q32856 | |
| Qubit dsDNA HS Assay Kit | Life Technologies | Q32851 | |
| Sequencer server system | Thermo Fisher Scientific | Torrent Suite Software | |
| Sequencing Reactions Universal Kit | Daan Gene Co., Ltd | 113 | https://www.daangene.com/pt/certificate.html This kit contains the following components: 1. Template Preparation Kit Set 1.1 Template Preparation Kit: Emulsion PCR buffer Emulsion PCR enzyme mix Template carrier solution 1.2 Template Preparation solutions: Template preparation reaction oil I emulsifier breaking solution II Template Preparation Reaction Oil II Nuclease-free water Tween solution Demulsification solution I Template washing solution C1 bead washing solution C1 bead resuspension solution Template resuspension solution 1.3 Template Preparation Materials: Reagent tube I connector Collection tube Reagent tube pipette I Amplification plate 8 wells strip Dedicated tips Template preparation washing adapter Template preparation filter 2. Sequencing Kit Set 2.1 Sequencing Kit: dGTP dCTP dATP dTTP Sequencing enzyme solution Sequencing primers Quality control templates 2.2 Sequencing Solutions: Sequencing solution II Sequencing solution IIII Annealing buffer Loading buffer Foaming agent Chlorine tablets C1 bead 2.3 Sequencing Materials: Reagent Tube II Reagent tube cap Reagent tube sipper II Reagent bottle sipper Reagent bottles 3. Chip |
| Sodium hydroxide solution | Sigma | 72068-100ML | |
| Thermal Cycler | Life Technologies | 4375786 |
References
- Driscoll, D. A., Gross, S. Clinical practice. Prenatal screening for aneuploidy. The New England Journal of Medicine. 360 (24), 2556-2562 (2009).
- Hassold, T., Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nature Reviews Genetics. 2 (4), 280-291 (2001).
- Hong, K. H., et al. Embryonic aneuploidy rates are equivalent in natural cycles and gonadotropin-stimulated cycles. Fertility and Sterility. 112 (4), 670-676 (2019).
- Adams, D. R., Eng, C. M. Next-generation sequencing to diagnose suspected genetic disorders. The New England Journal of Medicine. 379 (14), 1353-1362 (2018).
- Merriman, B., Team, I. T., Rothberg, J. M. Progress in ion torrent semiconductor chip based sequencing. Electrophoresis. 33 (23), 3397-3417 (2012).
- Quail, M. A., et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 13 (1), 341 (2012).
- Kane, S. C., Willats, E., Bezerra Maia, E. H. M. S., Hyett, J., da Silva Costa, F. Pre-implantation genetic screening techniques: Implications for clinical prenatal diagnosis. Fetal Diagnosis and Therapy. 40 (4), 241-254 (2016).
- Dilliott, A. A., et al. Targeted next-generation sequencing and bioinformatics pipeline to evaluate genetic determinants of constitutional disease. Journal of Visualized Experiments: JoVE. (134), e57266 (2018).
- Ion ReproSeq™ PGS View Kits User Guide. Thermo Fisher Scientific Available from: https://tools.thermofisher.com/contents/sfs/manuals/MAN0016158_IonReproSeqPGSView_UG.pdf (2017)
- PicoPLEX® Single Cell WGA Kit User Manual. Takara Bio USA Available from: https://www.takarabio.com/documents/User%20Manual/PicoPLEX%20Single%20Cell%20WGA%20Kit%20User%20Manual/PicoPLEX%20Single%20Cell%20WGA%20Kit%20User%20Manual_112219.pdf (2019)
- . Qubit® 3.0 Fluorometer User Guide, Invitrogen by Life Technologies Available from: https://tools.thermofisher.com/contents/sfs/manuals/qubit_3_fluorometer_man.pdf (2014)
- Ion AmpliSeq™ DNA and RNA Library Preparation User Guide. Thermo Fisher Scientific Available from: https://tools.thermofisher.com/contents/sfs/manuals/MAN0006735_AmpliSeq_DNA_RNA_LibPrep_UG.pdf (2019)
- Ion OneTouch 2 System User Guide. Thermo Fisher Scientific Available from: https://tools.thermofisher.com/contents/sfs/manuals/MAN0014388_IonOneTouch2Sys_UG.pdf (2015)
- Ion Pl Hi-Q OT2 200 Kit User Guide. Thermo Fisher Scientific Available from: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0010857_Ion_Pl_HiQ_OT2_200_Kit_UG.pdf (2017)
- Ion Pl Hi-Q Sequencing 200 Kit User Guide. Thermo Fisher Scientific Available from: https://tools.thermofisher.com/content/sfs/manuals/MAN0010947_Ion_Pl_HiQ_Seq_200_Kit_UG.pdf (2017)
- Torrent Suite Software 5.6. Help Guide. Thermo Fisher Scientific Available from: https://www.thermofisher.com/in/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-data-analysis-workflow/ion-torrent-suite-software.html (2017)
- Wiedenhoeft, J., Brugel, E., Schliep, A. Fast Bayesian inference of copy number variants using Hidden Markov models with wavelet compression. PLoS Computational Biology. 12 (5), 1004871 (2016).
- Rubio, C., et al. Pre-implantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials. Fertility and Sterility. 99 (5), 1400-1407 (2013).
- Gleicher, N., Kushnir, V. A., Barad, D. H. Preimplantation genetic screening (PGS) still in search of a clinical application: a systematic review. Reproductive Biology and Endocrinology. 12, 22 (2014).
- Bono, S., et al. Validation of a semiconductor next-generation sequencing-based protocol for pre-implantation genetic diagnosis of reciprocal translocations. Prenatal Diagnosis. 35 (10), 938-944 (2015).
- Handyside, A. H. 24-chromosome copy number analysis: a comparison of available technologies. Fertility and Sterility. 100 (3), 595-602 (2013).
- Wells, D., et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. Journal of Medical Genetics. 51 (8), 553-562 (2014).
- El-Metwally, S., Hamza, T., Zakaria, M., Helmy, M. Next-generation sequence assembly: Four stages of data processing and computational challenges. PLoS Computational Biology. 9 (12), 1003345 (2013).
- Jennings, L. J., et al. Guidelines for validation of next-generation sequencing-based oncology panels: A joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. The Journal of Molecular Diagnostics: JMD. 19 (3), 341-365 (2017).
- de Bourcy, C. F., et al. A quantitative comparison of single-cell whole genome amplification methods. PLoS One. 9 (8), 105585 (2014).
- Fiorentino, F., et al. Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical pre-implantation genetic screening cycles. Human Reproduction. 29 (12), 2802-2813 (2014).
- Damerla, R. R., et al. Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis. Mammalian Genome. 25 (3-4), 120-128 (2014).
- Brezina, P. R., Anchan, R., Kearns, W. G. Preimplantation genetic testing for aneuploidy: what technology should you use and what are the differences. Journal of Assisted Reproduction and Genetics. 33 (7), 823-832 (2016).
- Landrum, M. J., et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Research. 46, 1062-1067 (2018).
- Genomes Project, C, et al. A global reference for human genetic variation. Nature. 526 (7571), 68-74 (2015).
- McKusick, V. A. Mendelian inheritance in man and its online version, OMIM. American Journal of Human Genetics. 80 (4), 588-604 (2007).
- Wang, K., Li, M., Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Research. 38 (16), 164 (2010).
- Zhao, M., Zhao, Z. CNVannotator: A comprehensive annotation server for copy number variation in the human genome. PLoS One. 8 (11), 80170 (2013).
- Zhang, W., et al. Clinical application of next-generation sequencing in pre-implantation genetic diagnosis cycles for Robertsonian and reciprocal translocations. Journal of Assisted Reproduction and Genetics. 33 (7), 899-906 (2016).
- Xu, J., et al. Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human pre-implantation embryos. Proceedings of the National Academy of Sciences of the United States of America. 114 (41), 8695-8702 (2017).
