Next Generation Sequencing (NGS), why quantifying exact DNA/RNA is important
In addition, researchers have been provided with a new ability to probe genomes to a greater depth for a new understanding of genome sequence variants and how these underlie phenotype and disease. This is central to the new discipline of personalized medicine.
Next-generation sequencing (NGS) or high-throughput sequencing is a generic term to describe a number of different modern DNA/RNA sequencing technologies. These allow the sequencing of DNA and RNA to be carried out much quicker and less expensively than first generation Sanger sequencing.
Methods such as Illumina (Solexa) sequencing, Ion torrent: Proton / PGM sequencing, Illumina MiSeq and SOLiD sequencing have revolutionized genomic and genetic research.
Effectively there are two major models of next-generation sequencing (NGS) technology:
- short-read sequencing
- long-read sequencing
Short-read sequencing produces a large amount of data that researchers can use to investigate increasingly complex phenotypes, by maximizing the number of bases sequenced in the shortest time. These methods provide low-cost, higher-accuracy data useful for population-level research and clinical variant discovery.
Long-read sequencing aims to resolve structurally complex regions by sequencing long and continuous strands of DNA. These longer read length methods are better suited for de novo genome assembly applications and full-length isoform sequencing. This lends itself more to generating further in-depth data about genome structure and function.
There are a number of sequencing instruments on the market which use different technology. Illumina leads with benchtop machines such as the MiSeq, which is a fast, personal benchtop sequencer, with run times as low as 4 hours and outputs intended for targeted sequencing and sequencing of small genomes.
MiSeq sequencing uses clonal amplification of adopter-ligated DNA pieces on a glass slide surface. A cyclic reversible termination strategy is used to read the bases, by sequencing each nucleotide individually on the template strand, through successive rounds of base incorporation, washing, imaging and cleavage. The nucleotide is identified with fluorescent imaging.
Ion Torrent’s proton sequencing methodology uses an electronic lab-on-a-chip approach. pH changes induced by the release of hydrogen ions during DNA extension are measured by the semiconductor sequencing process. Detected pH changes are converted into a voltage signal by the sensor at the bottom of the microwell. The number of bases incorporated are proportional to the voltage signal.
The Single-Molecule Real-Time method of sequencing from Pacific Biosciences is another popular method. A capped template (SMRT-bell) is generated through ligation of single-stranded, hairpin adaptors onto the ends of digested DNA or cDNA molecules. This allows the DNA to be sequenced multiple times using a strand displacing polymerase, which increases accuracy.
Quantification and accuracy
Prior to sequencing, preparation of the sample is required: DNA is fragmented either enzymatically or mechanically and is afterwards ligated to adapters in order to mark the so-called “library” and to immobilize the fragments at the place of sequencing (e.g. glass slide for Illumina or beads used for IonTorrent). The adapters are specific to a library and hence report on the shared origin of sequences later. The fragments are then amplified and sequencing starts. The preparation of the DNA library is important for the success of sequencing.
Quantification of nucleic acids is required at (minimum) two stages of library preparation: input needs to be determined before fragmentation and just before sequencing. Optimized input amounts guarantee optimal fragmentation, avoids loss of rare material when using too low concentrations and inefficient fragmentation due to excessive material.
The pooling of libraries is basically the mix of different origin libraries. This is done because the capacity of modern NGS systems is higher than required for only one library. Operation at full capacity is only achieved by combining several libraries with accurately determined concentrations. These are pooled to result in the recommended optimal sequencing amount. Mistakes at this stage will undoubtedly intensify errors further downstream in the process, impacting on sequencing depth and coverage of sequencing.
The need to pool libraries underlines the throughput of NGS systems and explains the need for high throughput nucleic acid quantification.
High Throughput Scale of NGS
Next-generation sequencing (NGS) has altered the scale and throughput of work with nucleic acids. To ensure the smooth running of the high throughput system, the amount of nucleic acid in the starting material needs to be accurately quantified. Microplate readers allow scientists to accurately and rapidly quantify the levels of nucleic acids.
Traditionally nucleic acid is quantified either by absorbance or fluorescence. For absorbance quantification, the absorbance of nucleic acids at 260 nm is recorded. Acquisition of the whole UV-absorbance spectrum of a sample allows not only for DNA quantification, but also for identification of phenol and protein residuals which absorb at 230 nm and 280 nm, respectively. The measurement is often done on a microvolume spectrophotometer which requires only a microliter of sample, but can only measure one sample at a time. Hence, it is not compatible with high throughput.
Compared to UV absorbance, fluorescent DNA quantification is more sensitive, more specific and therefore recommended for NGS library preparation. Standard methods use a dye which becomes highly fluorescent in when associated with nucleic acids. A gold-standard for nucleic acid quantification for NGS purposes is Qubit™. The dye is mixed with a nucleic-acid containing sample and fluorescence is measured directly in the sample preparation tube. The fluorometer is limited to a single tube and therefore to low throughput.
Both nucleic acid quantification methods can be transferred to higher throughput by using a microplate reader and 96-, 384- or even 1536-well plates. BMG LABTECH microplate readers use ultrafast UV/Vis spectrometers for absorbance measurements. These combine speed and the acquisition of complete absorbance spectra making them ideal for nucleic acid quantification. In addition to microplates, the LVis plate holds up to 16 samples of two microliters and offers a low-volume but higher throughput solution for absorbance quantifications and qualifications of nucleic acids samples. Multi-mode readers can additionally measure the preferred fluorescent nucleic acid kits and as a consequence assume concentration and quality estimation by absorbance as well as sensitive and specific DNA measurement by fluorescence.
In summary, these are the advantages of using a microplate reader for nucleic acid measurements for NGS purposes:
- Higher throughput --> saves time
- Easily measure replicates --> higher significance and exact quantification
- One device for two methods --> less space and lower costs
- Possibility for assay miniaturization --> less sample volume and reduced costs
- Integration into automatic workflows --> less hands-on time and automated calculations
NGS technologies have evolved greatly since the Genome Project, leading to considerable improvements in yield and quality. Enhancements in chemistry, cost, throughput and availability are driving the rise of new, varied technologies to address applications that were not formerly possible. These include:
- integrated long-read and short-read sequencing studies
- routine clinical DNA sequencing
- real-time pathogen DNA monitoring
- enormous population-level projects
Although huge strides are being made in this technology, limitations remain, particularly for long homopolymer stretches and GC-rich regions where coverage and accuracy across the genome still have issues. From a technological perspective, the time required to sequence and analyse data, limits the use of NGS in clinical applications in which time is an important factor; the costs and error rates of long-read sequencing make it prohibitive for routine use, and ethical deliberations limit the public and private use of genetic data.
As well as these issues rapid analysis of future genomes will rely upon the development of standards for variant calling, data processing and reporting. Given the biases and limitations of individual sequencing platforms, accurate genome sequencing may also use multiple technologies. In the end for large-scale genomics to become fully accepted into a clinical environment, there is an absolute need to reduce the costs and timescales associated with storage and interpretation of genomic data.
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