Next Generation Gene Sequencing (NGS)

Posted by Safe Fertility Center is more common and less time consuming than the old technical tests. aCGH, SNPs and qPCR are common technologies that have been widely used in PGS worldwide. The potential application of PGS is rapidly extended by pathological genes of hereditary diseases, making it easier to obtain abnormal chromosomes based on sequence information.

The results of the Human Genome Project have had a huge impact on molecular biology and genetics in the 21st century. Many health problems have led to the search for important genes for both genetic and non-hereditary diseases, which has also led to the study of Sanger DNA sequencing technology. Sanger sequencing is the most commonly used technique for DNA sequencing, but the biggest problem with Sanger sequencing is the high cost and time-consuming testing.

Recently, new sequencing technologies have been partially used, and the problem of Sanger sequencing has been alleviated. Next-generation gene sequencing (NGS) has been introduced into the molecular sequencing field for 8 years.

The upgrade of next-generation gene sequencing technology has reduced the cost of each test, which has made the new molecular genetics field a hot research field. At present, clinical practice research requires a huge amount of these technologies, which presents unique challenges for experts, especially when NGS is used in medical laboratories.

Next-generation gene sequencing (NGS) is a more common and less time-consuming technique than the old ones. ACGH, SNPs and qPCR are common technologies that have been used worldwide for PGS. The potential application of NGSPGS is rapidly expanded by the pathogenesis of hereditary diseases, making it easier to obtain abnormal chromosomes based on sequence information.

NGS can produce a larger number of gene-sequence reads, covering a large number of bases per run (as shown in Figure 1). The three major platforms for NGS technology are currently in use, namely 454 /Roche, Solexa / Illumina and SOLiD/Life Technology (ABI). These platforms differ in sequencing order and the number of sequential read formats and read outputs. In addition, these platforms are quite effective in several different applications.

The platform form of Illumina (MiSeq) was selected as a tool to screen 24 abnormal chromosomes. Illumina's advanced Miseq technology for PGS testing makes it possible to detect gene translocation problems and single gene diseases before metastasis. In addition, with the application of new technologies, 24 chromosome assays can be used to screen for unbalanced chromosomes, including aneuploidy and embryonic chromosomal translocations that can be selected for implantation. Illumina's NGS technology not only detects the number of chromosomes, but also detects abnormalities after chromosomal rearrangements.

NGS can also provide chromosomal aberration testing, adding other genetic mutations and higher resolution data beyond the CNV (copy number variation) and AOH (no heterozygosity) functional chips. As functional resolution levels increase, NGS is an ideal way to measure chromosomal inversions, balance translocations, and disease-related point mutations.

VeriSeqTM PGS on the MiSeq® system (information from Illumina)

For PGS, the IVF next-generation gene sequencing solution used by the Millennium Reproductive Center provides accurate results for aneuploidy screening and expands opportunities for the future. The VeriSeq PGS solution, consisting of the VeriSeq PGS Kit – MiSeq and MiSeq systems, utilizes Next Generation Gene Sequencing (NGS) technology to provide a more comprehensive and accurate screening of all 24 chromosomes for euploid embryo selection. The effects of using VeriSeq PGS are comparable to the array-based 24sure technology that has been widely used. In addition, NGS helps improve inspection processes, increase throughput, and enhance performance.

Industry leading SBS chemistry offering the highest precision

VeriSeq PGS is based on the industry leading Illumina Synthetic (SBS) chemistry, which is the most widely used NGS technology. In fact, 90% of the world's sequencing data is generated using Illumina's technology. This proprietary, reversible, termination-based approach enables millions of DNA fragments to be sequenced in parallel and to detect single bases because they have been incorporated into the growing DNA strand. This method almost eliminates abnormalities and reactions associated with repeated nucleotide (homopolymer) strings. The overall accuracy of the homogenate depends on the length and the nucleotide composition of each region (adenine, thymine, guanine, or cytosine).

Illumina's sequencing covers the most accurate human genome, with the highest yields of error-free data reading, and the highest ratio of Q30* in the industry. Such high quality data greatly reduces the low false positive and false negative rates and reduces the large demand for downstream verification with reliable data support.

Fast, efficient workflow

Industry-leading data quality 90% of the world's sequencing data is generated using Synthetic Sequencing's Synthetic (SBS) technology

Fast, streamlined workflow. Sample results can be exported in approximately 12 hours

High yield analysis up to 24 samples per screening

Fast, efficient workflow

The VeriSeq PGS solution provides a fast, point-to-point PGS method in approximately 12 hours (Figure 1). Once the streamlined library is ready, use the VeriSeq DNA Library Kit to amplify the DNA. In the MiSeq system, the prepared library is placed in flow cells for screening. The instrument computer performs primary and secondary data analysis. The generated files will be imported into BlueFuse Multi-Software for analysis, data management and reporting of results.

Ultra low DNA input

After DNA is subjected to SurePlex DNA amplification, NGS requires as little as 1 nanogram of DNA to provide highly sensitive embryo screening. DNA can be obtained from embryonic leaf sections on the third day of embryos or a nourishing ectoderm (TE) biopsy from the blastocyst.

Increase production

With the use of NGS, users can analyze multiple samples simultaneously, which greatly increases production. Users can choose to process up to 24 fresh samples or up to 24 batches of frozen samples. This flexibility allows the laboratory to expand to meet future production needs (see Table 1).

Comprehensive data analysis and information management

VeriSeq PGS has a license for a complete solution that includes BlueFuse's multi-software, analysis and reporting of VeriSeq results. BlueFuse supports a complete laboratory workflow from receiving samples to reporting results (Figure 2).

Sample database

BlueFuse uses a scalable database architecture to store detailed information, experimental data and results for all samples. In addition, simple filters, powerful queries, and visualizing the performance of each IVF cycle ensure that the right information is available when needed. Within a BlueFuse multi-database, PGS data generated using the 24sure microarray can be analyzed, stored and viewed against VeriSeq PGS data.

BlueFuse multi-software

Accurate results from the VeriSeq PGS solution

Automatic analysis

The demultiplexed sample information is uploaded directly to the MiSeq system, saving time and allowing samples to be retrieved. Click on a shortcut to quickly access and run sample reports for quality control.

Clear profile

Powerful visualization capabilities generate profiles from thousands of flow cell measurement cells to fully understand the status and confirmed results of each chromosome.

Simple report

Complex calculations and showing normal or abnormal state of each chromosome, and include confidence estimates based on measured noise or any potential ambiguity. In addition to reproducibility and objectivity, it can also compare laboratory results with data published in the literature. The final product is an automated sample and cycle report.

Accurate aneuploidy screening

To demonstrate the utility of NGS for PGS, single cell data generated from the MiSeq system was compared to data generated by the 24sure assay, which is the most widely used PGS technique. For sequencing data, the number of sequences is a proportional copy number so that a larger or lower number of reads corresponds to tris or monomeric (Figure 3).


In a recent study, Fiorentino F, et al. (2014) used NGS to validate PGS3. In the blinded study, 18 single cells and 190 whole genome amplification (WGA) products consisting of a single blastomere were evaluated for 4,992 chromosomes, of which 402 also contained unbalanced copy numbers. . For aneuploidy (consistent response and assignment of chromosome copy number), the specificity of NGS was 99.98% at 100% sensitivity. For aneuploid embryos (24 chromosomes are consistently diagnosed), the specificity of NGS is 100% at 100% sensitivity. The positive and negative predictive values based on the NGS-24 chromosome aneuploidy screening protocol were 100%, respectively.

look forward to

Introducing NGS into the lab to demonstrate the beginning of a new opportunity for PGS. As the application covers the entire human genome, NGS has opened up new testing products that have led to a gradual increase in portfolios and products.

summary

VeriSeq PGS Kit – The MiSeq and MiSeq systems introduce NGS into IVF, increasing the likelihood of improving pregnancy success rates. Accurate aneuploidy screening results are comparable to current industry standards, but this is only the beginning of NGS. With more and more in-depth research, NGS opens up new opportunities and enables more accurate performance of improved workflows.


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