Regarding better feasibility and less time consuming for testing than previous techniques, aCGH, SNPs and qPCR are the common technology that has been widely use for PGS worldwide. The potential applications of PGS have been expanded dramatically with the easy availability of sequence-based information on chromosomal abnormalities through pathologic genes responsible for inherited diseases. The achievement of human genome project has a great impact on the molecular biology and genetics in 21st century. The state of art of Sanger DNA sequencing is driven the research through many health problems for seeking human genes that are crucial for inherited and non-inherited diseases. Sanger sequencing method has been remained the most common technology for DNA sequencing techniques, however the most problems relevant to Sanger technique are cost and time consuming of the test. Recently sequence technique has been partially supported and reduced the problems from Sanger sequencing, the next-generation sequencing was introduced to the molecular sequencing area for last 8 years. Growing up of new evolution as next-generation sequencing with decline trend of cost per assay are the main properties that make new interesting technology for coming up explosion of new molecular genetic field. Now, there is considerable demand to apply these technologies for research-to-clinical practice that brings with unique set of challenges, particularly when it come to setting up NGS in the medical laboratory.
Next generation sequencing (NGS) technology can achieve much quickly and efficiently read the underlying sequence of an organism by means of massively parallel sequencing than Sanger’s technology.
NGS can produce much larger number of sequence reads that cover huge number of bases per run (as shown in figure1). Three major platforms of NGS technologies are currently in used, namely 454/Roche, Solexa/Illumina and SOLiD/Life Technology (ABI). These platforms differ in sequencing principles, sequence read formats and number of read outputs. Moreover, these platforms are useful in the different applications in several approaches.
The platform form of Illumina (MiSeq) is selected as a tool to screening 24-chromosome abnormalities. The advanced of Miseq for PGS test from Illumina is possible to detect translocation and single-gene disorder before transfer. In addition, 24 chromosomal detection under new technologies can be used to screen the imbalance of chromosome including aneuploidy and chromosomal translocation of the embryo s that can be selected for implantation. Not only the number of chromosome that can be detected, the abnormal of rearrangement of its can be done using NGS from Illumina.
NGS offers complimentary chromosome aberration testing that can add additional mutational and higher-resolution data beyond the CNV (Copy number variation) and AOH (absence of heterozygosity) capabilities of microarrays. With higher levels of functional resolution, NGS is ideal for measuring chromosome inversions, balanced translocations, and disease-associated point mutations.
VeriSeqTM PGS on the MiSeq® System (Information from Illumina)
A next-generation sequencing solution for PGS, providing accurate results in aneuploidy screening and extending future opportunities.
The VeriSeq PGS solution, consisting of the VeriSeq PGS Kit – MiSeq and the MiSeq System, takes advantage of next-generation sequencing (NGS) technology to provide comprehensive, accurate screening of all 24 chromosomes for selecting euploid embryos. PGS results generated using VeriSeq PGS are comparable to those achieved with the widely used array-based 24sure technology. In addition, NGS offers the opportunity for improved assay workflow, higher throughput, and enhanced performance.
Industry-Leading SBS Chemistry Delivers Highest Accuracy
VeriSeq PGS relies on the industry-leading Illumina sequencing by synthesis (SBS) chemistry, the most widely adopted NGS technology. In fact, 90% of the world’s sequencing data are generated using Illumina technology. This proprietary reversible terminator-based method enables massively parallel sequencing of millions of DNA fragments, detecting single bases as they are incorporated into growing DNA strands. The method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers). The accuracy across homopolymers can vary depending on the length and the nucleotide composition of each region (abundance of adenine, thymine, guanine, or cytosine).
Illumina sequencing delivers the most accurate human genome at any coverage, the highest yield of error-free reads, and the highest percentage of base calls above Q30* in the industry. Such high data quality results in low false positive and false negative rates, reducing the need for extensive downstream validation while providing full confidence in the data.
Fast, Efficient Workflow
The VeriSeq PGS solution offers a fast, end-to-end PGS method that is completed in about 12 hours (Figure 1). It begins with DNA extraction and amplification from a single embryonic cell using the SurePlex DNA Amplification Kit. Amplified samples undergo streamlined library preparation using the VeriSeq DNA Library Kit. Prepared libraries are loaded onto a flow cell for sequencing on the MiSeq System. An on-instrument computer performs primary and secondary data analysis. Generated files are imported into BlueFuse Multi software for analysis, data management, and results reporting.
Ultra-Low DNA Input
NGS offers a highly sensitive method for screening embryos, requiring as little as 1 ng of DNA from a SurePlex DNA amplification reaction. DNA can be obtained from a blastomere biopsy from a day 3 embryo or from a trophectodermal (TE) biopsy from a blastocyst.
With NGS, user can multiplex samples for simultaneous analysis, greatly increasing throughput. Choose to process up to 24 fresh samples, or batch up to 24 frozen samples. This flexibility enables laboratories to begin scaling up today to meet future throughput needs (Table 1).
Comprehensive Data Analysis and Information Management
VeriSeq PGS includes a license for BlueFuse Multi software, a complete solution for analyzing and reporting VeriSeq results. BlueFuse supports the complete laboratory workflow, from sample receipt to results software (Figure 2).
BlueFuse uses a scalable database architecture to store all sample details, experimental information, and results. Simple filters, powerful queries, and visual representation of each IVF cycle make sure that the right information is available when needed. Within a BlueFuse Multi database, PGS data generated using 24sure microarrays can be analyzed, stored, and viewed alongside VeriSeq PGS data.
Demultiplexed sample information is uploaded directly from the MiSeq System, saving time and allowing sample tracking. Single-click shortcuts provide rapid access to run and sample reports for easy QC.
Powerful visualization capabilities generate profiles from thousands of pooled measurements from each flow cell, enabling full understanding of the status of each chromosome and results confirmation.
Sophisticated algorithms calculate and call the status for each chromosome as either normal or abnormal, and include an estimate of confidence in the call based on assay noise or any underlying ambiguity. In addition to reproducibility and objectivity, this enables comparison of laboratory results with those published in the literature. The end product is an automated sample and cycle report.
Accurate Aneuploidy Screening
To demonstrate the utility of NGS for PGS, data generated from single cells on the MiSeq System was compared to data from a 24sure assay, the most widely used technology for PGS.
For sequencing data, the number of sequences is proportional to copy number so a greater or lower number of reads will correspond to trisomy or monosomy (Figure 3).
In a recent study, Fiorentino F, et al. (2014) validate use of NGS for PGS3. In a blinded study, 18 single cells and 190 whole-genome amplification (WGA) products from single blastomeres were evaluated, assessing 4,992 chromosomes, 402 of which carried a copy number imbalance. NGS specificity for aneuploidy call (consistency of chromosome copy number assignment) was 99.98% with a sensitivity of 100%. NGS specificity for aneuploid embryo call (24-chromosome diagnosis consistency) was 100% with a sensitivity of 100%.
Both positive and negative predictive values of the NGS-based 24-chromosome aneuploidy screening protocol were 100%.
Bringing NGS into the laboratory for PGS represents the beginning of new opportunities. With applications spanning the entire human genome, NGS opens up new assay offerings, enabling an incremental increase in portfolio offerings.
The VeriSeq PGS Kit – MiSeq and MiSeq System bring the power of NGS to IVF, providing the potential to increase pregnancy success rates. The accurate aneuploidy screening results are comparable to the current industry standards. But this is just the beginning for NGS.
As we learn more, NGS opens up improved workflows for more precise performance and new opportunities.
1. Scott RT Jr, Ferry K, Su J, Tao X, Scott K, et al. (2012) Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: a prospective, blinded, nonselection study. Fertil Steril 97(4): 870–875.
2. Tobias E, Connor JM, Ferguson-Smith (2011) Essential medical genetics. 6th edition: 243–247. Chichester, West Sussex, UK. Wiley-Blackwell.
3. Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, et al. (2014) Development and validation of a next-generation sequencing–based protocol for 24-chromosome aneuploidy screening of embryos. Fertil Steril