Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Summary

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

Abstract

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

Introduction

Aneuploidy is the abnormality in the number of chromosomes by the presence of one or more extra chromosomes or the absence of one or more chromosomes. Embryos that carry some type of aneuploidy, such as the loss of one X chromosome (Turner syndrome), extra copies of autosomes, like trisomies of autosome 21 (Down syndrome), 13 (Patau syndrome), and 18 (Edwards syndrome), or extra sex chromosomes such as 47, XXY (Klinefelter syndrome) and 47, XXX (Triple X syndrome), can survive to term with birth defects¹. Aneuploidy is the primary cause of first trimester miscarriages and in vitro fertilization (IVF) failure². I....

Protocol

All protocols and the trophectoderm (TE) biopsy (1.1.1.1 section) applied in this study were reviewed and approved by the human research ethics committee of No. 924 hospital on September 18th, 2017 (NO: PLA924-2017-59). The patients/participants provided their written informed consent to participate in this study.

1. DNA isolation from human embryo biopsy and whole genomic amplification

1. Protocol for whole genome amplification^9,

Discussion

Chromosomal aneuploidy of embryos is the cause of a large proportion of pregnancy loss, whether conceived naturally or in vitro fertilization (IVF). In the clinical practice of IVF, it is proposed that screening the embryo aneuploidy and transferring the euploidy embryo could improve the outcome of IVF. Fluorescence in situ hybridization is the earliest technique adopted for sex selection and PGT-A; however, this technique requires more technical expertise from laboratory personnel and is relatively lab.......

Materials

Name	Company	Catalog Number	Comments
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