Microfluidic whole genome haplotyping
Encyclopedia
Microfluidic
whole genome haplotyping is a technique for the physical separation of individual chromosomes from a metaphase cell followed by direct resolution of the haplotype
for each allele.
basis. Current methods of next generation sequencing
are capable of identifying heterozygous loci, but they are not well suited to identify which polymorphisms exist on the same (in cis) or allelic (in trans) strand of DNA. Haplotype information contributes to the understanding of the potential functional effects of variants in cis or in trans. Haplotypes are more frequently resolved by inference through comparison with parental genotypes, or from population samples using statistical computational methods to determine linkage disequilibrium between markers.
Direct haplotyping is possible through isolation of chromosomes
or chromosome segments. Most molecular biology
techniques for haplotyping can accurately determine haplotypes of only a limited region of the genome.
Whole genome direct haplotyping involves the resolution of haplotype
at the whole genome level, usually through the isolation of individual chromosomes.
A haplotype
(haplo: from Ancient Greek
ἁπλόος (haplóos, “single, simple”) is a contiguous section of closely linked segments of DNA
within the larger genome
that tend to be inherited together as a unit on a single chromosome
. Haplotypes have no defined size and can refer to anything from a few closely linked loci up to an entire chromosome
. The term is also used to describe groups of single-nucleotide polymorphisms (SNPs) that are statistically associated.
Most of the knowledge of SNP association comes from the effort of the International HapMap Project
, which has proved itself a powerful resource in the development of a publicly accessible database of human genetic variation.
Phasing
is the process of identifying the individual complement of homologous chromosome
s. Methods for phasing include pedigree analysis, allele-specific PCR, linkage emulsion PCR haplotype analysis, polony
PCR, sperm typing, bacterial artificial chromosome
cloning, construction of somatic cell hybrids, atomic force microscopy, among others. Haplotype phasing can also be achieved through computational inference methods.
refers to the use of micro-sized channels on a micro-electro-mechanical system (MEMS
). Microfluidic channels have a diameter of 10-100μm, making it possible to manipulate and analyze minute volumes. This technology combines engineering, physics, chemistry, biology, and optics. Over the past decades it has revolutionized micro and nanoscale biology, genetics and proteomics. Microfluidic devices can combine several analytical steps into one device. This technology has been coined by some as the "lab on a chip" technology. Most current molecular biology methods use some form of MEMS, including microarray
technology and next generation sequencing
instruments.
device.
cell is isolated from solution. The chromosomes are then released from the nucleus, and the cytoplasm is digested enzymatically. Next, the chromosome suspension is directed towards multiple partitioning channels. The chromosomes are physically directed into the partitioning channels using a series of valves. In the first description of this technique, Fan et al. designed a custom-made program (MatLab) to control this process. Once separated, the chromosomes are prepared for amplification by sequential addition and washout of trypsin, denaturation buffer and neutralization solution. The DNA is then ready for further processing. Because of the small amount of DNA, amplification needs to be performed using kits specialized for very small initial DNA quantities. The amplified DNA is flushed out of the microfluidic device and solubilized by the addition of a buffer. The amplified DNA can now be analyzed by various methods.
Once the chromosomes have been isolated and amplified any molecular haplotyping can be applied as long as the chromosomes remain distinct. This could be accomplished by keeping them physically separated, or identifying each sample by genotyping. Once each chromosome has been identified each pair of homologs can be assorted into one of two haploid genomes.
, prenatal diagnosis
and in the characterization of cancer cells.
Whole genome haplotyping through microfluidics will increase the rate of discovery within the HapMap project, and provides an opportunity for corroboration and error detection within the existing database. It will further inform genetic association studies.
As methods for amplification of small amounts of DNA improve, single chromosome sequencing is possible using microfluidics to separate each individual chromosome. A cost-effective approach may be to barcode each individual chromosome and perform parallel resequencing of the entire individual genome. The amplification of each chromosome separately also provides a mechanism to potentially fill in some of the gaps that remain in the human reference genome
. Single chromosome sequencing will allow for unmapped sequences to be associated with a single chromosome. Additionally, single chromosome sequencing will be more accurate in the identification of copy number variants and repetitive sequences.
. Single cell analysis does not account for the possibility of mosaicism; therefore, applications in cancer diagnosis and research would necessarily require processing of multiple cells. Finally, since this entire process is based on amplification from a single cell, the accuracy of any genetic analysis is limited to the ability of commercially available platforms to produce sufficient amounts of unbiased and error free amplicon.
is another process for isolating single chromosomes for genetic analysis. As with the above technique microdissection begins with metaphase cells. The nucleus is lysed mechanically on a glass slide and part of the genetic material is partitioned under microscope. The actual microdissection of genetic material was initially accomplished through the careful use of a fine needle. Today computer-directed lasers are available. The genomic area isolated can range from part of a single chromosome, up to several chromosomes. To accomplish whole genome haplotyping the microdissected genomic section is amplified and genotyped or sequenced. Like with the microfluidic technique, specialized amplification platforms are necessary to address the problem of a small initial DNA sample.
library into various pools of equal size presents an alternative method for haplotype phasing. In the proof of principle description of this technique 115 pools were created containing ~5000 unique clones from the original fosmid library. Each of these pools contained roughly 3% of the genome. Between the 3% in each pool and the fact that each clone is a random sampling of the diploid genome, 99.1% of the time each pool contains DNA from a single homolog. Amplification and analysis of each pool provide haplotype resolution limited only by the size of the fosmid insert.
http://www.phgfoundation.org/news/7134/
Microfluidics
Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
whole genome haplotyping is a technique for the physical separation of individual chromosomes from a metaphase cell followed by direct resolution of the haplotype
Haplotype
A haplotype in genetics is a combination of alleles at adjacent locations on the chromosome that are transmitted together...
for each allele.
Whole genome haplotyping
Whole genome haplotyping is the process of resolving personal haplotypes on a whole genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
basis. Current methods of next generation sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
are capable of identifying heterozygous loci, but they are not well suited to identify which polymorphisms exist on the same (in cis) or allelic (in trans) strand of DNA. Haplotype information contributes to the understanding of the potential functional effects of variants in cis or in trans. Haplotypes are more frequently resolved by inference through comparison with parental genotypes, or from population samples using statistical computational methods to determine linkage disequilibrium between markers.
Direct haplotyping is possible through isolation of chromosomes
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
or chromosome segments. Most molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
techniques for haplotyping can accurately determine haplotypes of only a limited region of the genome.
Whole genome direct haplotyping involves the resolution of haplotype
Haplotype
A haplotype in genetics is a combination of alleles at adjacent locations on the chromosome that are transmitted together...
at the whole genome level, usually through the isolation of individual chromosomes.
Haplotype
A haplotype
Haplotype
A haplotype in genetics is a combination of alleles at adjacent locations on the chromosome that are transmitted together...
(haplo: from Ancient Greek
Ancient Greek
Ancient Greek is the stage of the Greek language in the periods spanning the times c. 9th–6th centuries BC, , c. 5th–4th centuries BC , and the c. 3rd century BC – 6th century AD of ancient Greece and the ancient world; being predated in the 2nd millennium BC by Mycenaean Greek...
ἁπλόος (haplóos, “single, simple”) is a contiguous section of closely linked segments of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
within the larger genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
that tend to be inherited together as a unit on a single chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
. Haplotypes have no defined size and can refer to anything from a few closely linked loci up to an entire chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
. The term is also used to describe groups of single-nucleotide polymorphisms (SNPs) that are statistically associated.
Most of the knowledge of SNP association comes from the effort of the International HapMap Project
International HapMap Project
The International HapMap Project is an organization that aims to develop a haplotype map of the human genome, which will describe the common patterns of human genetic variation. HapMap is a key resource for researchers to find genetic variants affecting health, disease and responses to drugs and...
, which has proved itself a powerful resource in the development of a publicly accessible database of human genetic variation.
Phasing
Phasing
Haplotype
A haplotype in genetics is a combination of alleles at adjacent locations on the chromosome that are transmitted together...
is the process of identifying the individual complement of homologous chromosome
Homologous chromosome
Homologous chromosomes are chromosome pairs of approximately the same length, centromere position, and staining pattern, with genes for the same characteristics at corresponding loci. One homologous chromosome is inherited from the organism's mother; the other from the organism's father...
s. Methods for phasing include pedigree analysis, allele-specific PCR, linkage emulsion PCR haplotype analysis, polony
Polony
Polony is a contraction of "polymerase colony," a small colony of DNA.Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase...
PCR, sperm typing, bacterial artificial chromosome
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...
cloning, construction of somatic cell hybrids, atomic force microscopy, among others. Haplotype phasing can also be achieved through computational inference methods.
Microfluidics
MicrofluidicsMicrofluidics
Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
refers to the use of micro-sized channels on a micro-electro-mechanical system (MEMS
Microelectromechanical systems
Microelectromechanical systems is the technology of very small mechanical devices driven by electricity; it merges at the nano-scale into nanoelectromechanical systems and nanotechnology...
). Microfluidic channels have a diameter of 10-100μm, making it possible to manipulate and analyze minute volumes. This technology combines engineering, physics, chemistry, biology, and optics. Over the past decades it has revolutionized micro and nanoscale biology, genetics and proteomics. Microfluidic devices can combine several analytical steps into one device. This technology has been coined by some as the "lab on a chip" technology. Most current molecular biology methods use some form of MEMS, including microarray
Microarray
A microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
technology and next generation sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
instruments.
Principle
Direct deterministic phasing of individual chromosomes can be achieved by isolating single chromosomes for genetic analysis through the use of a microfluidicMicrofluidics
Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
device.
Methods
A single metaphaseMetaphase
Metaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
cell is isolated from solution. The chromosomes are then released from the nucleus, and the cytoplasm is digested enzymatically. Next, the chromosome suspension is directed towards multiple partitioning channels. The chromosomes are physically directed into the partitioning channels using a series of valves. In the first description of this technique, Fan et al. designed a custom-made program (MatLab) to control this process. Once separated, the chromosomes are prepared for amplification by sequential addition and washout of trypsin, denaturation buffer and neutralization solution. The DNA is then ready for further processing. Because of the small amount of DNA, amplification needs to be performed using kits specialized for very small initial DNA quantities. The amplified DNA is flushed out of the microfluidic device and solubilized by the addition of a buffer. The amplified DNA can now be analyzed by various methods.
Once the chromosomes have been isolated and amplified any molecular haplotyping can be applied as long as the chromosomes remain distinct. This could be accomplished by keeping them physically separated, or identifying each sample by genotyping. Once each chromosome has been identified each pair of homologs can be assorted into one of two haploid genomes.
Applications
Microfluidic direct deterministic phasing allows all the chromosomes to be isolated in the same experiment. This unique feature suggests possible applications within clinical, research and personal genomics realms. Some of the possible clinical applications for this technique include phasing of multiple mutations when parental samples are unavailable, preimplantation genetic diagnosisPreimplantation genetic diagnosis
In medicine and genetics pre-implantation genetic diagnosis refers to procedures that are performed on embryos prior to implantation, sometimes even on oocytes prior to fertilization. PGD is considered another way to prenatal diagnosis...
, prenatal diagnosis
Prenatal diagnosis
Prenatal diagnosis or prenatal screening is testing for diseases or conditions in a fetus or embryo before it is born. The aim is to detect birth defects such as neural tube defects, Down syndrome, chromosome abnormalities, genetic diseases and other conditions, such as spina bifida, cleft palate,...
and in the characterization of cancer cells.
Whole genome haplotyping through microfluidics will increase the rate of discovery within the HapMap project, and provides an opportunity for corroboration and error detection within the existing database. It will further inform genetic association studies.
As methods for amplification of small amounts of DNA improve, single chromosome sequencing is possible using microfluidics to separate each individual chromosome. A cost-effective approach may be to barcode each individual chromosome and perform parallel resequencing of the entire individual genome. The amplification of each chromosome separately also provides a mechanism to potentially fill in some of the gaps that remain in the human reference genome
Reference genome
A reference genome is a digital nucleic acid sequence database, assembled by scientists as a representative example of a species' genetic code. As they are often assembled from the sequencing of DNA from a number of donors, reference genomes do not accurately represent the genetic code of any...
. Single chromosome sequencing will allow for unmapped sequences to be associated with a single chromosome. Additionally, single chromosome sequencing will be more accurate in the identification of copy number variants and repetitive sequences.
Limitations
As of January 2011, only one publication has described use of this technique. The scientific commons awaits further validation of this method and its efficacy in isolating and amplifying analyzable amounts of DNA. While this method does streamline the process of chromosome isolation, certain parts in the process – such as the initial isolation of a metaphase cell – remain difficult and labour intensive. Other automated techniques for metaphase cell separation would improve throughput. In addition, this method is only applicable to cells in metaphase, which inherently limits the technique to cell types and tissues that undergo mitosisMitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
. Single cell analysis does not account for the possibility of mosaicism; therefore, applications in cancer diagnosis and research would necessarily require processing of multiple cells. Finally, since this entire process is based on amplification from a single cell, the accuracy of any genetic analysis is limited to the ability of commercially available platforms to produce sufficient amounts of unbiased and error free amplicon.
Chromosome microdissection
Chromosome microdissectionChromosome microdissection
Chromosome microdissection is a technique that physically removes a large section of DNA from a complete chromosome. The smallest portion of DNA that can be isolated using this method comprises 10 million base pairs - hundreds or thousands of individual genes....
is another process for isolating single chromosomes for genetic analysis. As with the above technique microdissection begins with metaphase cells. The nucleus is lysed mechanically on a glass slide and part of the genetic material is partitioned under microscope. The actual microdissection of genetic material was initially accomplished through the careful use of a fine needle. Today computer-directed lasers are available. The genomic area isolated can range from part of a single chromosome, up to several chromosomes. To accomplish whole genome haplotyping the microdissected genomic section is amplified and genotyped or sequenced. Like with the microfluidic technique, specialized amplification platforms are necessary to address the problem of a small initial DNA sample.
Large insert cloning
Randomly partitioning a complete diploid fosmidFosmid
Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids are 40 kb of random genomic DNA...
library into various pools of equal size presents an alternative method for haplotype phasing. In the proof of principle description of this technique 115 pools were created containing ~5000 unique clones from the original fosmid library. Each of these pools contained roughly 3% of the genome. Between the 3% in each pool and the fact that each clone is a random sampling of the diploid genome, 99.1% of the time each pool contains DNA from a single homolog. Amplification and analysis of each pool provide haplotype resolution limited only by the size of the fosmid insert.
External links
International HapMap Project Web Sitehttp://www.phgfoundation.org/news/7134/