6.3 Genotyping

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What is genotyping?

 

Genotyping is a technique used to assay specific genes in an individual's genome in order to determine which allelic variations are present.  It is on a smaller scale than whole genome sequencing and the alleles interrogated are compared to a reference sequence.  Whole genome sequencing is now relatively fast and gives researchers and clinicians a full view of a person of interest's genome.  It is often assumed that genome sequencing has made the comparatively more established genotyping techniques obsolete.  However, genotyping is still highly relevant.  

As of 2011, the cost of high-throughput sequencing was approximately $0.0000003 per base using an industry standard 454 approach (1). The diploid human genome is approximately 6 billion base pairs long. So to sequence a single human genome, the approximate cost would be $1800. While sequencing is much more affordable than when Sanger sequencing was state-of-the-art, it may not be the most practical option for all projects.  Sequencing 1,000 individuals for a study is a significant cost, and genotyping is cheap.

 

Genotyping is also considered more reliable than high-throughput sequencing. For a method to be adopted into clinical laboratory practice in the United States it must meet the standards of the Clinical Laboratory Improvement Amendment (CLIA) (2). This quality control protocol is warranted. If a sequencer is accurate for each base pair it sequences 99.99% of the time and a 6 billion base pair genome is sequenced, the sequencer makes ~600,000 errors for one genome.  Conversely, genotyping only tests select and small number of sites, and therefore, produces fewer errors will per run compared to sequencing.

 

 

Genome Sequencing

Genotyping

Appropriate applications

More appropriate for large projects, e.g. discovering genome-wide associations among genes unknown to have a role in a ovarian cancer..

More cost-effective for smaller, more targeted projects, e.g. identifying variants known to increase cancer risk in an individual by probing select SNPs.  

Cost per run

More expensive per run
~$2,500-$8,000 for 454 sequencing (5)

Less expensive per run, ~$100-500 per pre-fabricated chip, depending on number of SNPs and platform (6)

Cost per base/SNP

Less expensive per base

$0.0000003 (1)

More expensive per SNP, varies extraordinarily depending on platform (6,7)
 

Amount of data

Enormous amount of data requiring bioinformatic analysis

Manageable amount of data to analyze, but also limited to information at specific SNPs

Requires reference genome or knowledge of sequence

No, though it is helpful

Yes, requires knowledge of the sequence to create probes

High throughput

Yes

No

Table 6.3.1. Differences between genome sequencing and genotyping.

 

 

The general method for single nucleotide polymorphism (SNP) genotyping (3)

 

Genotyping traditionally involves three key steps. The first is carrying out a polymerase chain reaction (PCR), amplifying segments of DNA which are of interest. The second step is then to distinguish between alleles for the segments of interest. There are many different ways to do this; some methods exploit hybridization qualities, polymerase interactions, ligation, variable primer extension and pyrosequencing. The different methods produce products that are distinguishable from one another depending on the genetic code of the individual being tested. The final step in this process is detecting the differences. This can also be done in many ways, such as mass spectrometry, fluorescent tagging, absorbance and melting temperature. Below, we highlight two methods, both of which are used in genotyping for research or clinical purposes today: 1) steady state hybridization and 2) Golden gate genotyping assay.

 

 

1) The steady state hybridization method [3]

 

The steady state hybridization method takes advantage of the ability of nucleic acids to hybridize to corresponding sequences more strongly for sequences with higher complementarity.  DNA samples are first amplified using PCR, and then labelled with a signal, usually using deoxyribonuclic tri-phosphates (dNTPs) that are fluorescently tagged in the PCR step. A microarray with sequence probes attached is then dipped into the sample solution, and subsequently rinsed to get rid of the excess PCR products. The microarray is redundant for sequences of interest, where different sequences for different alleles of the same gene are all spotted on it. The microarray is then exposed to increased temperatures, to the point where sequences without a perfect match will tend to denature. The excess sample is then rinsed off again. The melting temperature range which discriminates the perfectly matched sequences versus the imperfect ones can be increased using different nucleic acid analogs, such as locked nucleic acid analogs. The microarray is then analyzed by detecting which areas of the array display fluorescence, signifying which probes the sample stayed bound to when exposed to the increased temperatures. Generally, a computer will be programmed to interpret the results.

 

 

2) The GoldenGate genotyping assay (4)

 

Illumina's GoldenGate assay is a more recent innovation in genotyping that has allowed for a far larger number of SNPs to be analyzed in one run. Like the steady state hybridization method, it utilizes the concept of matching sequences; however, it also incorporates the use of DNA polymerase and ligase to produce differentiated signals according to the SNP of interest. First, the DNA samples of interest are activated using paramagnetic binding molecules. The assay requires that for every SNP locus, there exist three specific oligonucleotides which are added to the solution. Two types of these are called allele-specific oligonucleotides (ASOs), and these directly hybridize to the SNP of interest. The two types of ASOs possess a variable nucleotide which will represent the two possible matching nucleotides for the SNP. This nucleotide is located at the ASO's 3' end. To elaborate, say that one site is polymorphic for an A and a G, then the two types of ASOs will have a T or a C at the variable site. The last of the three oligonucleotides is the locus-specific oligonucleotide (LSO). The LSO binds a segment of DNA 1-20 base pairs down of the polymorphic site. The ASOs have different primer binding sites at their 5' ends according to the type of nucleotide they possess at their variable site. All LSOs have the same primer at their 3' ends. The oligonucleotides are allowed to hybridize to the sample DNA, and then DNA polymerase and ligase are added to the solution. DNA polymerase fills the gap of sequence between the ASOs and LSOs, and DNA ligase ligates the two separate strands together if the hybridization between allele is made with the appropriate ASO variable nucleotide site at the 3' end. The solution with ASOs and LSOs linked by the newly made sequence is then subjected to PCR using primers which are differentially labeled according to which ASO they bound. In addition, primers that bound LSOs would have a specific sequence that would localize them to a specific bead on the chip. The PCR products are mounted on a bead chip and then analyzed according to the wavelengths which each emit; the different ASOs providing different wavelengths of light to distinguish which SNPs the sample possessed. The patterns of wavelengths as they show on the bead chips are analyzed by a computer.

 

 

References

  1. Sawyer, E. High throughput sequencing and cost trends. Scitable. NatureEducation. July 13, 2011. http://www.nature.com/scitable/blog/bio2.0/high_throughput_sequencing_and_cost. Accessed March 10, 2013.
     
  2. Centers for Disease Control and Prevention. (April 5, 2012). Clinical Laboratory Improvement Amendments. Retrieved from: http://wwwn.cdc.gov/clia/default.aspx
     
  3. Chen, X., Sullivan, P. (2003). Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput. The pharmacogenomics journal. 3: 77-96.
     
  4. Shen, R., Fan, J., Campbell, D., Chang, W., Chen, J., Doucet, D., Yeakley, J., Bibikova, M., Wichham Garcia, E., McBride, C., Steemers, F., Garcia, F., Kermani, B., Gunderson, K., & Oliphant, A. (2005). High-throughput SNP Genotyping on universal bead array. Mutation research/fundamental molecular mechanisms of mutagenesis, 573(1-2): 70-82
     
  5. Polymorphic DNA Technologies, Inc. Roche 454 DNA Sequencing - with PCR prep and library prep options. Retrieved from: http://www.polymorphicdna.com/roche.html
     
  6. CCHMC Genetic Variation and Gene Discovery Core Facility.  SNP GENOTYPING.  Retrieved from: http://dna.chmcc.org/www/snpgen_main.php
     
  7. Partners HealthCare Center for Personalized Genetic Medicine (PCPGM).  Genotyping Pricing.  Retrieved from: http://pcpgm.partners.org/research-services/genotyping/pricing