A new era of genome sequencing technologies began in 2003, marked by the completion of the Human Genome Project using Sangers method. Since then, the diversity and volume of sequenced genomes continues to rise and the cost per megabase (106 bases) has continued to decrease.
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Furthermore, researchers are now able to probe genomes to a greater depth which enables new understanding of genome sequence variants and how they underlie disease and phenotype. These advances are critical in the new discipline of personalized medicine.
High-throughput sequencing or next-generation sequencing (NGS) is a term generically used to describe various modern DNA/RNA sequencing technologies. These mean that DNA and RNA can be sequenced much more quickly and also less expensively than first generation Sanger sequencing.
Methods that have revolutionized genomic and genetic research include: Illumina (Solexa) sequencing, Ion torrent: Proton/PGM sequencing and Illumina MiSeq and SOLiD sequencing.
NGS Strategies
There are two major models of next-generation sequencing (NGS) technology:
- Long-read sequencing
- Short-read sequencing
The aim of long-read sequencing is to resolve structurally complex regions by sequencing long, continuous strands of DNA. Longer read length methods are best suited to full-length isoform sequencing and de novo genome assembly applications. This means it can generate further detailed data about genome function and structure.
Short-read sequencing produces a large amount of data which can be used by researchers to investigate increasingly complex phenotypes. This is done by maximizing the number of bases sequences in the shortest time. These methods provide higher-accuracy, low-cost data which is useful for clinical variant discovery and population-level research.
NGS Methodology
There are various sequencing instruments on the market which use different technology. Illumina leads with machines such as the MiSeq: a fast, personal benchtop sequencer that has run times as low as 4 hours and outputs intended for sequencing of small genomes and targeted sequencing.
MiSeq sequencing utilizes clonal amplification of adopter-ligated DNA pieces on a glass slide surface. To read the bases, a cyclic reversible termination strategy is used which sequences each nucleotide individually on the template strand, through successive rounds of base incorporation, washing, imaging and cleavage. Fluorescent imaging is used to identify the nucleotide.
An electronic lab-on-a-chip approach is used in Ion Torrent’s proton sequencing methodology. The semiconductor sequencing process measures pH changes induced by the release of hydrogen ions during DNA extension. The sensor at the bottom of the microwell converts detected pH into a voltage signal. The voltage signal is proportional to the number of bases incorporated.
Another popular method is the Single-Molecule Real-Time method of sequencing from Pacific Biosciences. Ligation of single-stranded, hairpin adaptors onto the ends of digested DNA or cDNA molecules generates a capped template (SMRT-bell). The means that a strand displacing polymerase can be used to sequence the DNA multiple times and this 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
The scale and throughput of work with nucleic acids has been altered by next-generation sequencing (NGS). The amount of nucleic acid in the starting material needs to be accurately quantified to allow the smooth running of the high throughput system. Scientists are able to accurately and rapidly quantify the levels of nucleic acids using microplate readers.
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 Microplate 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
Conclusion
Since the Genome Project, NGS technologies have greatly evolved and this has led to considerable improvements in quality and yield. Improvements in cost, chemistry, availability and throughput are driving the rise of new, varied technologies to address applications that have not been possible before.
These include:
- Routine clinical DNA sequencing
- Enormous population-level monitoring
- Integrated long-read and short-read sequencing studies
- Real-time pathogen DNA monitoring
Despite the huge strides being made in this technology, limitations remain, particularly for GC-rich regions and long homopolymer stretches where coverage and accuracy across the genome are still problematic. From a technological standpoint, the time required to sequence and analyze 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 use routinely and the public and private use of genetic data is limited by ethical deliberation.
Additionally, rapid analysis of future genomes will rely upon developing standards for variant calling, data processing and reporting. Accurate genome sequencing may also use multiple technologies given the biases and limitations of individual sequencing platforms. There is an absolute need to reduce the costs and timescales associated with storage and interpretation of genomic data in order for large-scale genomics to become fully accepted into a clinical environment.
References
- Sanger F, Nicklen S, Coulson AR, DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7.
- Goodwin, Sara; McPherson, John D.; McCombie, W. Richard (17 May 2016). "Coming of age: ten years of next-generation sequencing technologies". Nature Reviews Genetics. 17 (6): 333–51.
- Grada A (August 2013). "Next-generation sequencing: methodology and application". J Invest Dermatol. 133 (8): 1–4.
- Schuster SC (January 2008). "Next-generation sequencing transforms today's biology". Nat. Methods. 5 (1): 16–18.
- Quail MA, Smith M, Coupland P, et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012;13:341.
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