SNP Testing | Contexo.Info

About SNPs

The Human Genome Project and subsequent 1000 Genomes Project have shown that the sequence of DNA nucleotides from one person to the other varies little. In fact, it is estimated that all humans share about 99.9% identical DNA. To avoid the redundancy and expense of excessive whole genome sequencing, scientists are using a spot check approach to learn about the impact of the differences in the remaining .01%.

Genome sequencing projects have identified millions of single base pair locations on human chromosomes that are polymorphic (poly = variable; morph = form) among humans. The base pairs at those locations (or loci) are called single nucleotide polymorphisms (SNPs).

What are SNPs?

23andme (18 Apr 2012) Genetics (Part 2 of 5): What are SNPs? [Video file] retrieved from https://youtu.be/tJjXpiWKMyA

Each SNP (usually pronounced snip) represents a difference in a single nucleotide pair. For example, one persons may have the base pair T-A at a specific location on chromosome 16 and another may have the base pair C-G in the same location. To be classified as a SNP, the variation must occur in at least 1% of the population.

Less common variations, those that occur in less than 1% of the population, are called single nucleotide variants or SNVs. In genealogy discussion boards, presentations and blogs you may also hear SNVs called private SNPs. The promise of SNVs as individual family markers is being embraced by genetic genealogists with optimism. Private SNPs are especially promising for Y chromosome surname projects where there is a need to characterize and sort individual lines of descent from a common male line ancestor. There are roughly 10 million SNPs spread throughout the human genome. They occur about once in every 300 nucleotides and are most commonly found in regions between genes as opposed to within protein coding or regulatory regions.

A SNP arises as a mistake or mutation during DNA replication. On extremely rare occasions, as DNA polymerase moves along a growing nucleotide chain, it mistakenly inserts the wrong nucleotide pair and a SNV results. If that happens during the process of meiosis, the SNV will be passed down to the offspring. From there, the SNV may be passed to future generations and eventually become common enough in the population (>1%) to be classified as a SNP. With 3 billion base pairs in the human genome, the likelihood of the same mutation occurring at the same locus more than once is extremely low. In fact, it is thought that SNPs are so rare that each human SNP has occurred only once in human history. For example, the SNP that is responsible for all the blue eyes in the world is thought to have occurred only once. All blue eyed people in the world descend from that person.

When SNPs do fall within coding or regulatory regions of DNA, they may play a direct role in the expression of traits or diseases that are affected by that gene’s function. Genome Wide Association Studies (GWAS) have been successful in finding genetic variations that contribute to such diseases as asthma, cancer, diabetes, heart disease and mental illness.

Most SNPs are functionally neutral. They have no effect on traits, health or development. Nevertheless, those SNPs are proving to be very useful as biological markers for nearby genes. Such SNPs may predict risks of developing certain diseases, responses to drugs, and susceptibilities to toxins.

Because SNPs are inherited they are also very useful for defining or verifying family lines and relationships. Ancestry Informative Markers (AIMs) are SNPs that differ in allele frequencies across different populations. Most such variations are shared by members of the same population so a high frequency of that allele will be a characteristic of the population. These SNPs are used in ethnicity predictions to distinguish between people of different geographic and cultural backgrounds.

There are many many more SNPs to explore!

Early methods of characterizing DNA were expensive, time consuming and limited in scope. The Sanger Sequencing method was used for most of the Human Genome Project. Over the years, Sanger Sequencing has undergone repeated updates until it is now a highly-automated process that can handle as many as 96 samples of test DNA at a time. Sanger Sequencing is still the gold standard for DNA sequencing. It does not readily lend itself to efficient SNP typing.

In the last ten years or so dramatic advances in technology have made it possible to analyze thousands of different DNA segments simultaneously. There are many variations of these new technologies and many more in development. As a group, they are frequently referred to as Next Generation or Next Gen. Next Generation technologies are especially well suited for SNP characterization.

Next Generation technologies are based on the natural ability of DNA to hybridize. A single strand of DNA nucleotides (ssDNA) can hybridize (match up and form a double strand held together by hydrogen bonds) with any other ssDNA that has a complementary nucleotide sequence. This is true even if the two strands are NOT from the same source.

Next Generation technologies typically bind millions of short oligonucleotide fragments (short chains of nucleotides) of known sequences to microscopic dots or minuscule silicon beads on the surface of a small glass or silicon slide. These fragments, called oligos, serve as probes than can hybridize to fragments of unknown DNA.

It is then possible to infer the nucleotides of the unknown DNA strand using base pairing rules and knowledge of the nucleotide sequence of the probe.

There are a variety of ways microarray chips can be designed, produced and used depending on research goals and objectives. The three major US companies that provide direct to the consumer DNA SNP testing for genealogy are AncestryDNA, Family Tree DNA and 23andme. All three use some version of the Illumina OmniExpress Chip.

Figure 1 Infinium OmniExpress-24 Kit

To create the Illumina OmniExpress BeadChip, single stranded DNA fragments of known sequences are bound to microscopic silicon beads. Each bead is only 3 microns in diameter (1 micron = 0.001 millimeter). Wells that are 3 microns deep are etched into the surface of the chip. A mixture containing beads with all the sequences to be examined is flooded onto the chip. The beads randomly settle into the wells where they are held tightly by physical forces.

All DNA fragments on a single bead are identical sequences of 50 or so oligonucleotide pairs that are also identical to a sequence adjacent to one of the SNPs that is to be assayed. The fragments are fashioned from sequences acquired from libraries of known DNA segments of the human genome. They are amplified by PCR using primers designed to select the exact sequences for each SNP. Each sequence begins with a unique identifying address of 29 nucleotides at its 5' end.

Figure 2 By Philippe Hupé [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

Beads for each sequence are prepared separately. Then all the beads for the SNPs to be tested are mixed in equal amounts before being flooded onto the surface of the chip. Each sequence to be studied is represented by about 20 beads in different places in the microarray. This insures consistent and accurate data for each SNP.

The known oligonucleotide sequences on each bead will serve as reference sequence for the test DNA fragment that binds with it. However, the beads are deposited randomly during chip fabrication. Before the chip can be of any use, it must be determined which probes are in which wells. The company providing the chip supplies a digital file with this information to the customer at the time of delivery. This decoding is done by performing a series of hybridizing reactions between the address segments of the probes and fluorescence labeled decode oligonucleotides. More information about the decoding process can be found in Gunderson, Kevin L. et al. “Decoding Randomly Ordered DNA Arrays.” Genome Research 14.5 (2004): 870–877. PMC. Web. 25 June 2017.

As miniaturized technologies have enabled smaller and smaller bead and well size, the number of beads that can be adhered to one chip has increased and consequently the number of SNPs that can be evaluated by one chip has increased. Currently the chips being used to assay SNPs for genealogical purposes accommodate from 700,000 to 1,000,000 segments for and can assay 24 different samples at once.

Designing and producing a DNA microarray chip requires much time and expertise. Fortunately, there are commercial companies that offer “off the shelf” human DNA chips designed for a variety of research purposes. A large lab that does a lot of specialized work can collaborate with these companies to design custom chips with SNPs specific for their research objectives. AncestryDNA, 23andme and Family Tree DNA all use custom designed chips from Illumina. They are all similar and their results can be compared using third party services but each includes some proprietary SNPs that are unique to the individual company’s focus

As a hypothetical, overly simplified example of the SNP typing process using an Illumina OmniExpress microarray bead chip, assume you want to determine the allele for a SNP on human chromosome 16 that is present as T-A in most humans but as G-C in a substantial minority (>1%). Since chromosomes are paired—one maternal and one paternal in origin, there are three possible allele combinations for each individual. They are illustrated below.

The more prevalent TA combination has been inherited from both parents. It will be present on both chromosomes. The individual is homozygous for TA at that SNP.

The TA allele has been inherited from one parent and the less common CG from the other. The SNPs will differ on the two chromosomes. The individual is heterozygous for that SNP.

Both parents passed the less common CG allele. The SNPs on both chromosomes are the same. The individual is homozygous for CG at that SNP.

The more prevalent TA combination has been inherited from both parents. It will be present on both chromosomes. The individual is homozygous for TA at that SNP.

The TA allele has been inherited from one parent and the less common CG from the other. The SNPs will differ on the two chromosomes. The individual is heterozygous for that SNP.

Both parents passed the less common CG allele. The SNPs on both chromosomes are the same. The individual is homozygous for CG at that SNP.

Figure 1 The thee possible combinations of the SNP allele variants TA and CG. The top two strings of letters represent the SNP and surrounding nucleotides on one of the chromosome pair 16 and the bottom two strings represent the same segment on the other chromosome 16.

Preparing the Microarray

The first step in the procedure is to prepare beads with nucleotide sequences that are identical to the known sequences next to the SNP in chromosome 16. That nucleotide sequence must be designed to stop precisely at the base just before the SNP.

Because only the nucleotides of the actual SNP are different, the area surrounding the SNP on chromosome 16 will be the same for both genotypes TA and CG. The SNP is generically represented by the ? in bold blue font:

Figure 2 The SNP and its surrounding nucleotides

The probe segment will be the sequence of nucleotides to the left of the SNP:

Figure 3 The probe

Segments from the part of chromosome 16 where the SNP resides can be acquired from a library of human genome segments. The segments are processed by PCR with primers designed to identify and amplify that precise segment adjacent to but not including the SNP. A 29 base segment identifying sequence, an address, (shortened and represented in purple below) is added to one end of the fragment during this time. The result will be thousands of fragments of the probe sequence with the address one end:

Figure 4 The address plus the probe

The fragments are then immobilized by binding them to the microscopic silicon beads. The beads are flooded onto the chip where they settle into the prepared wells.

Though this example includes only one SNP, a single chip will accommodate thousands of beads each with thousands of copies of one oligonucleotide sequence designed to correspond to a region adjacent to one of the 700,000 to 1,000,000 SNPs to be analyzed.

5’ ACTCTACTAACTGGA?CAATGCGTACGT 3’
3’ TGAGATCATTGACCT?GTTACGCATGCA 5’
Figure 2 The SNP and its surrounding nucleotides

The probe segment will be the sequence of nucleotides to the left of the SNP:

5’ ACTCTACTAACTGGA__ 3’
3’ TGAGATCATTGACCT__ 5’
Figure 3 The probe

5’ GATCACTCTACTAACTGGA__ 3’
3’ CTAGTGAGATCATTGACCT__ 5’
Figure 4 The address plus the probe

Finding the Genotype of the SNP

DNA from saliva or a cheek cell scrape is extracted and purified. Using PCR, the segments are fragmented and amplified. Primers are designed to flank the SNP and all the bases corresponding to those in the oligo fragments on the chip. This insures the sample fragments will be able to find matching complementary sequence on the chip. Using a variety of specially designed primers makes it possible to isolate DNA segments for many SNPs at once.

The solution containing the isolated and purified DNA fragments that include the SNP is flooded over the chip. The chip is then heated to denature the DNA strands (cause them to separate) on the beads and in the test solution. As the temperature is then cooled, free floating, single strands of the test DNA drift through the cell and are attracted by their single stranded complements on the beads. A substantial number of the test fragments will anneal (bond) to the probe fragments.

At this point a portion of the immobilized DNA segments are double stranded, hybridized nucleotide segments with one strand from the test DNA and one from the probe DNA. There is one exposed base on the test strand. This is because the probe strand was designed to stop one base pair short of the SNP.

Figure 5 The probe DNA has bound to the test sequence and immobilized it on the chip.

5’ ACTCTACTAACTGGA?CAATGCGTACGT 3’
3’ TGAGATCATTGACCT?GTTACGCATGCA 5’
Figure 5 The probe DNA has bound to the test sequence and immobilized it on the chip.

Not all the free floating single stranded DNA will hybridize with a probe so the chip is washed to remove free floating DNA.

Then it is flooded with a mixture of free dinucleotide bases that have been labeled with fluorescent molecules. Typically, the complementary bases adenine and thymine are labeled to fluoresce red and guanine and cytosine green. Recall from Sanger Sequencing that dideoxynucleotides can bind to a growing DNA strand at its 5’ end. However, the 3’ end has been altered to block the addition of another nucleotide to the chain. DNA polymerase extends the probe side of the DNA segment one more base by adding the complementary base to the exposed SNP base

Figure 6 The ddNTP molecules are washing around the immobilized probe and template.

The chip is washed again to remove unused ddNPTs.

Then is is scanned with red and green lasers.If the test sample has an A or T at the SNP position, DNA polymerase will have added a fluorescence labeled A or T which will make the bead glow read when it is illuminated by ultraviolet light. If the sample has a G or C in the SNP position, it will have been matched with its fluorescent complement and will glow green.

Figure 7 The chip is glowing red. Either an A or a T has been added to the probe chain.

The bead has glowed red reporting that the probe chain has added either an A or a T. From that, we know that the nucleotide pair at the SNP is AT.

Figure 7 portrays three possible outcomes for a SNP with two different alleles.

Figure 7

Both chromosomes carry the more common allele TA. The individual is homozygous for the SNP

One chromosome carries the more common allele TA and the other carries the less common allele CG. The individual is heterozygous for the SNP.

Both chromosomes carry the less common allele CG. The individual is homozygous for the SNP.

Since humans are diploid, we have two versions of each SNP. If both SNP’s are A, then all the beads for that SNP will glow red. Similarly, if the test subject is homozygous for G, all the beads for the SNP will glow red. However, if the test subject is heterozygous for the SNP, he or she will have one allele that is A and one that is T. Some of the oligos will glow red and some green. Together red and green light make yellow so beads from someone who is heterozygous will glow yellow.

Figure 8

About SNPs

What are SNPs?

Preparing the Microarray

Preparing the Microarray

Finding the Genotype of the SNP

Finding the Genotype of the SNP

Where Can I Go From Here?

Where Can I Go From Here?