AMERICAN SOCIETY FOR NUTRITION SYMPOSIUM/WORKSHOP Genetic polymorphisms as sources of nutritional/metabolic variation – a methods workshopBasic laboratory techniques and quality control Werner G. Bergen• PhD, The Ohio State University• Prof. Emeritus, Michigan State University• Professor, Biochemistry, Cell & Molecular Biosciences / Animal Sciences, Auburn University• Research: Molecular regulation of lipid metabolism in large animal models• Instruction: - Principles of Biochemistry (UG)- Advanced Lipid Biochemistry (G)

AMERICAN SOCIETY FOR NUTRITION SYMPOSIUM/WORKSHOP

Genetic polymorphisms as sources of nutritional/metabolic variation – a methods workshopBasic laboratory techniques and quality control – a

primer.

Werner G. Bergen and Jacek Wower

Cell and Molecular Biosciences/Animal Sciences

Auburn University, AL

Auburn released its 2011 football schedule Tuesday.

Topics

• In this workshop this presentation is about laboratory techniques relevant to genomics/ GWAS.

• Here, I will limit my discussion to:

• Nucleic acid isolation .

• Genotyping platforms.

• Detection systems for SNP.

• What is downstream from here?

Overarching Issues in Adopting Technologies for Genomic Analysis

• Sample preparation/purification

• Platform/System for SNP interrogation

• Actual Chemistries/ Procedures to be used

• Sample preparation (ie. DNA and RNA isolation) is mostly under the purview of the investigator .

• Platform or system adopted will relate to the specific aims of the research. There are many choices of kits, protocols, equipment; choices here depend on actual needs and fit into investigator’s research program.

• Procedures and platforms are constantly evolving as well.

• Overall success then is related to sample preparation and ability to utilize the procedures (often kits); platform development may be useful for comparative studies where species specific “tools” are not available.

Core bench techniques for gene (DNA) polymorphisms/GWAS

• Genomic DNA isolation

• Genotyping (SNP)

• Sequencing & fine mapping and variations thereof as necessary

• PCR applications

• Next steps: Data analysis, bioinformatics, computational biology, statistical evaluation (not discussed here).

DNA isolation• DNA/nucleoproteins are precipitated and the DNA extracted with alkali. Since

RNA are acid (precipitant) soluble this is essentially a RNA free DNA (Schmidt-Thannhauser ). Not used much directly, but basis for nucleic isolation procedures.

• DNAzol ®. Tissue is lysed in a guanidine-detergent solution and the DNA is precipitated with ethanol. Following an ethanol wash, DNA is solubilized in water or 8 mM NaOH.

• ChargeSwitch®gDNA (invitrogen) is a high speed , PCR ready DNA isolation application kit. Here the DNA is harvested from a lysis system by positively charged beads/plates and released by raising the pH; any co-isolated RNA can be removed with Rnase. This appoach uses no strong reagents and chaotropic salts (There are many other such kits for research and forensic use).

• Other contemporary procedures involve cell lysis with detergents , proteases followed by adsorption to silica columns. All non bound reagents, salts and cellular constituents are removed. Finally the DNA is eluted from the silica column (favor DNA binding) for downstream applications. Residual RNA may be removed with Rnase.

• All such procedures have been adapted for high-through-put, robotics technologies.

• To conduct high quality genomics research the quality of the starting nucleic acid material is of great concern.

RNA isolations

• Trizol®. Single step, guanidine isothiocyanate-phenol-chloroform: Mostly employed for RNA including RNA representing <200bp.

• Column silica matrix adsorption: Typically in kits (These are either DNA and RNA specific).

• Beads kits.

• In RNA isolation, the proof is in the “outcome”.

• Major issues: Fragility, chemical contamination from isolation reagents, degradation, DNA contamination, yield issues.

• Sample stabilizers..RNA later etc…

• Practice and maintain strict adherence to quality standards: These basically include fragmentation analysis with denaturing agarose gel electrophoresis or capillary electrophoresis (Bioanalyzer to obtain RNA integrity values) and spectroscopy to obtain 260/280nm ratios.

• Thus to obtain high quality RNA, procedures must be diligently followed. This starts with tissues/ cells sample handling through all steps in the procedures.

The Trizol® method for nucleic acid isolation

Schroeder et al., 2006 BMC Molec Biol, 7:3

RNA Integrity Values. RIN greater than 8 needed for micro-array analysis, RIN greater at 6 qRT-PCR still possible.

Agilent Bioanalyzer 2100

Ling Tang, PhD 2006, Auburn University

“A big disappointment”

Genotyping

• Genotyping is the process of determining the genes (genotype) of an individual by examining the individual's DNA sequence.

• SNP genotyping is the measurement of genetic variation of single nucleotide polymorphisms (SNPs) between members of a species

• Based on knowledge of DNA/gene sequences, prior validations , ID of SNP/alleles; utilize specific primers or array probes.

• Variety of platforms available for small and modest SNP numbers

• For large number of SNPs: Use an array (hybridization) format approach which can entail over 1 M SNP per sample. Depending on circumstances such assays may be best conducted in a University Core/ Service center or a commercial laboratory. High quality genomic DNA is still critical here.

Some common characteristics of all genotyping systems• Approaches: Methods are adaptations of “multiple molecular biology

techniques” including primer synthesis, hybridizations (W-C), utilization specific enzymes, PCR and post amplification methods.

• All systems/platforms have probes /primers that are designed for certain SNP within the genome (Chromosomal number & location; some platforms may have very high numbers of such SNP specific probes and quality control probes). Unknown SNP not detected.

• SNP detection systems include: simple base extension, allele specific extension, primer extension, ligation dependent, hybridization dependent specific molecular probes, differential hybridization, high resolution melting approaches.

• SNP detections utilize the concept of misalignment or missing complementary bases to pinpoint SNP sites.

• Various unique, proprietary methods (kits), and software to mark SNPs. Some sequencing/re-sequencing SNP detection approaches are now coming into more frequent use.

Use of genotyping techniques

• Diagnostics

• Single target to few genes association studies

• Global association studies

• Pooling of samples (should be limited to candidate genes identification)

A Partial List of Technology Platforms for Genotyping

Vendor Platform Detection*

• Illumina Infinium-Beads Hapten labeled Primer- extension, antibody

• Affymetrix High density array Gene Chip Scanner

• Seqenom MassARRAY (medium) Primer extension-MS- TOF

• Qiagen TaqMan/PCR 5’exonuclease-fluorophore

• Post-PCR High Melting Resolution (HMR)

• PCR/gels Specific primers Agarose gels/ amplicon sizes

Sequencing, other detection kits, capillary

electrophoresis

• There are many platform/detection specific “chemistries”

Flow of a Genotyping Experiment Isolation of DNA

Choosing desired genotyping procedure

Set-up and optimize

Run genotyping experiment

Utilize platform associated software for raw data capture

Data normalization, quality control, bio-statistical analysis

Some SNP identification systems

Allele-specific cleavage in an Invader® reaction by flap endonucleases (FENs).

Detect SNP; methods may include primer extension-fluorescent dyes, stains, dye termination, high resolution melting approaches, capillary electrophoresis

Cluster Analysis of SNP dataQiagen Fast-SNP PCR Kit

GWAS strategies• Traditional: explorations of the whole genome in a

subset of subjects to define a most appropriate SNP panel (Phase 1).

• Then in phase 2 a more restricted SNP panel will be used for GWAS.

• With the availability of lower costs total genome coverage, it can be argued that the power of these explorations can be increased by identifying all SNP in the larger subject pool. Here the likelihood of missing less frequent SNP will be lowered and overall data robustness will be enhanced.

Approaches and platforms for GWAS SNP determinations.From: Edenberg and Liu 2009; Cold Spring Harbor Protocols. Doi:10.1101/pdb.top62

Some other bench level issues related to genotyping

• It is critical that contaminants be eliminated (may overestimate DNA) and/or interfere with genotyping and sequencing.

• gDNA amplification may lower “power” to detect less frequent SNP

• Limited target DNA may result in poor array type genotyping • SNPs for which many samples give no genotype may be missed

• May require retesting and re-sequencing .

An example of a novel SNP technique in identifying HPV virus integration into human DNA.

From: Multiplex PCR capillary electropherogram of pappillomavirus genotype integration into human DNA. Infectious Agents and Cancer (open access) 6:1, 2011

Newly available approaches in next-generation GWAS

Almost daily new or refined approaches are available for improved “bench level work”

These include for example Roche NimbleGen services/products*:

Sequence capture

DNA copy number differences

Chip-chip-DNA (cis) protein binding domains (promoters)

DNA methylation high resolution technology

Pathway gene expression systems (SA-Qiagen)

Comparative Genome SequencingThis is a highly competitive market between a number of manufacturers. Products will be either for sale or offered as services

* No products or services are endorsed.

Affymetrix “The Way Ahead” *

• Slice variant analysis• Exon scanning• Whole transcriptome analysis• Studies of regulatory mechanisms• WGAS (here already)

• Other critical concerns:

• Next-generation sequencing (it is here)• Epigenetic analyses and influence on GWAS• miRNA influences on GWAS _________________________________

• No product is being endorsed

Summary

• Overall strategies in nutritional sciences research are changing from a “nutrition experiment-sample analyses” paradigm using randomly allocated experimental subjects to strategies also incorporating genomic level aspects in our nutrition research. Knowledge is generally lacking on how individual genetic variations/polymorphisms effect nutrient requirements and utilization, metabolism and nutritional excess or deficiencies associated disease processes. I have reviewed here some basic aspects of the “bench level” involvement in genomics/GWAS studies to address this subject. Underlying my comments was the point that downstream efforts, no matter how sophisticated, are highly dependent on upstream diligence.

Good Luck

Illumina (latest sequencing equipment) Roche

AffymetricsSpotted array

Liquid nitrogen cooled mortarsand pestles

Contemporary Life Sciences, GWAS, Nutrition and Genomics and the Bench

• This workshop was undertaken to focus on forefronts of nutrition research and to explore the impact of specific new sets of tools on nutrition research; today I have discussed some basic aspect of the “bench level” involvement in genomics and GWAS. Underlying my comments was the point that downstream efforts, no matter how sophisticated, are highly dependent on upstream diligence.

• We are now entering with full force genomic level studies to understand nutrition, metabolism , disease processes etc. Exciting new data are emerging from either DNA based analyses (genotyping, SNPs, gene locations), global gene expression analysis (RNA) (and proteomics) and an ever bourgeoning array of bioinformatics tools to interpret our data. Outcome of these efforts will affect how we practice our science in the future and what we do as nutritional scientists. Remember in the past nutrition research efforts were primarily bench based; contemporary research will use multiple approaches. Techniques such as global assessments of biological processes and metabolites followed by bioinformatics based raw data assessment and interpretation appear to have changed how we will conduct research in nutrition today and in the future.

A compendium of ever evolving methods being applied in genomics (GWAS) and functional genomics research (examples)

• Arrays, expression, structure

• Second generation sequencing

• Micro-fluidic Chips

• Chromatin immuno-precipitation/ protein-DNA interactions

• Other DNA-protein studies

• Targeted Genotyping and Sequence Enrichment Tools (some industry leaders believe that discovery will be achieved more with arrays than sequencing tools). With rapidity of progress in sequencing this may not turn out.

• SNP from the 1000 Genomes Project

• Illumina’s HumanOmni2.5 Quad DNA Analysis (and others newer platforms)

• Genome-wide microarrays provide a global view of copy variant studies, but fine mapping and validation studies still require much more targeted approaches

• DNA methylation (epigenetics) and miRNA assessments

Epicentre Biotechnologies

Epicentre Biotechnologies

Signosis’ array®Example of 232 miRNA array

Approaches and platforms for GWAS SNP determinations.From: Edenberg and Liu 2009; Cold Spring Harbor Protocols. Doi:10.1101/pdb.top62

• Bench Level Genomic and Functional Genomics Research includes:

• Tissue nucleic acid extraction and quality control

• DNA based procedures, standard PCR

• Genotyping /SNP/ Molecular diagnosis

• High throughput PCR and Second Generation Sequencing

• Genomic level to Transcriptomic level

• Role of expression/ RNA based procedures/microarrays/ tissue cell heterogeneity

• Epigenetics, mutation analysis, copy number variations

• Non-coding, regulatory RNA (a new[er] emerging paradigm)

• In this talk we will focus on some contemporary technologies with the recognition that technologies & applications are always changing

DNA RNA Protein

An example of an SNP technique in identifying/genotyping HPV

integration in human DNA. • Highly specific primers for conserved exons and short amplicons were

utilized to genotype 21 potentially oncogenic HPV in a primer rich multiplex-single tube PCR system.

• Primer construction provided pairs of primers to obtain amplicons separated in size by ~3 or more nt to maintain high PCR efficiency (amplicon size denotes conserved exons for multiple genotypes imposed by primer design).

• All reverse primers were synthesized with 5’ labels ( FAM, blue; HEX, green or NED, black alternatively with each increase in amplicon size).

• DNA was extracted from appropriate tissues

• Multiplex PCR were run and amplicons were separated by capillary electrophoresis

• Data was analyzed with GeneMarker software and conserved exons for six HPV were found in in the DNA.

Expression (RNA) based genome association studies, metabolic patterns and pathway analyses

• Experiments and treatments

• Samplings (tissues, body fluids) & physiological measurements

• Plasma metabolite analysis (possibly metabolomics), body composition, weight gain, other observations

• Tissue processing and RNA isolation

• Expression analysis by microarrays, pathways arrays or marker/target genes (RT-PCR, analysis platforms) for certain pathways (global to very specific)

• Statistics and Bioinformatics/analysis (this area is really advancing)

• Integrated analysis: Obtain transcriptomic signatures, map expression data on metabolic networks, pathway analyses etc.

• Interpretations/implications

Genotyping

• Approaches: Hybridization based, Enzyme Based, Post amplification based on physical properties of DNA methods.

• Candidate gene studies; examples are: can test for1-48 SNPs in each sample. Systems based on genomic DNA and assays that measure individual SNP to low level multiplexing of 10 SNP, to an oligonucleotide ligation assay to discriminate SNPs followed by PCR (SNP specific primers ) and capillary amplicon analysis (up to 48 SNP) intermediate number SNP assays . Also may use pyro-sequencing and single primer PCR systems. All these procedures are available in kits and can be adapted to “on site” laboratories (more later).

• Large number of SNPs: Use an array (hybridization) format approach which can entail over 1 M SNP per sample (and also copy number values). Unless one has a large well equipped (with highly trained technical personnel) laboratory, available assays may be best conducted in a University Core/ Service center or a commercial laboratory. High quality genomic DNA is still critical here

Key components of a “typical” GWAS study (DNA linked)• High SNP coverage either from existing genotyping or new

data. Important points here are genome coverage, distance between SNP, chromosomal position. Also role of location of candidate genes near SNPtag/trait loci can be assessed (Linkage disequilibrium and haplotype associations). These are not “bench issues, but these and more are considerations for the useful application of genotyping (construction of SNP arrays, primer choices).

• Results tabulations or SNP calls.

• Measuring relevant traits (growth, rate of fattening, response to nutrient deficiency, metabolomics).

• Statistically determine associations between SNP, linkage disequilibrium and phenotypic characteristics. Stringent statistical procedures, avoid biases.

• Biological interpretations; validations, eventually from associations to causal relationships.

Developments and improvements of specific kits is highly competitive between various manufacturers. Here is a very recent advertisement comparing Qiagen’s Rneasy with the Ambion Pure link RNA mini kit. (Remember this is an Ambion ad)

We would like for example to relate variations in nutritional needs, efficiencies and deficiencies to genetic polymorphism (DNA and functional protein) for both genome wide scans of genes and also for smaller empirical nutrition studies on more specific target gene(s) polymorphisms and alleles.

Increased understanding is needed about relationships between nutrient needs, efficiency of nutrient use and both single gene (Mendelian) and polygenic related polymorphisms at the genomic level, particularly as a primary regulatory mechanisms for function or dysfunction in nutrition and metabolism.

Background/Preface: Goals Related to this Workshop

Preface (continued)

Nutrient requirements for example may be established by empirical feeding studies relating intake of a nutrient to a response and/or sufficiency criteria. In inbred rats such studies provide “requirement” values with low variations while in wild type breeding animals the “requirement “ values may differ widely and it is more difficult here to pinpoint a requirement value. In such cases safety factor corrections (such as a daily dietary allowance or +/- 1, 2 SD) have been applied to ensure that all organisms within a population receive adequate nutrition. We would like to know more about the genomic basis of variations in nutrient use etc.

In another example some of the drug metabolizing enzymes for warfarin, losartin etc and also endocrine disruptors may vary in their efficiency (eg V and/or S at ½ Vm). Such differences in enzymatic activity have been shown to be related to alleles and consequent differences in catalytic potential of enzymes (0-100%). In metabolic processes depending on multiple enzymes (polygenic traits), multiple alleles of rate limiting enzymes may affect nutrient use, deficiencies as well as disease.

Flow of a “typical” GWAS study (DNA linked)• DNA isolation and subsequent SNP detections with various

procedures or platforms (specificity is achieved in the assay system which may include certain primers, microarray type probes etc).

• Capturing raw data and assessment driven by platform specific software

• Results tabulations ; often initial clustering of homozygotes and heterozygotes.

• Measuring relevant traits (growth, rate of fattening, response to nutrient deficiency, metabolomics enzyme activities).

• Statistically determine associations between SNP, linkage disequilibrium and phenotypic characteristics. Stringent statistical procedures, avoid biases.

• Biological interpretations; validations, eventually from associations to causal relationships.

Some Technical Issues in Nucleic Acid Isolation

• RNA acid soluble, DNA bound very tightly to basic proteins alkali soluble. Basis of classical Schmidt-Thannhauser and Schneider procedures to separate DNA and RNA (circa 1945). These approaches provided total DNA and RNA content but not DNA and RNA for downstream applications. Nucleic acids precipitate in ethanol (solvents).

• Need to get rid of protein contamination, guanidinium thiocyanate, phenol, salts and other rapid protein precipitants for downstream applications.

• There are seem to be nucleases everywhere

• Single step isolation (eg Trizol; chaotropic salts/ phenol/ chloroform) good yields but possible reagent carryover without downstream purification

• Kits: guanidinium salts precipitation-enzyme (Rnase) inactivation, cell lysis, silica or membrane adsorption, lower yields -but likely less reagent carryover

• To obtain high quality RNA, procedures must be diligently followed. This starts with tissues/ cells sample handling through all steps in the procedures.

Possible sites of SNP/polymorphisms in a single gene

5’ +1 TSS 3’

TATA

Upstream Core promoter UTR Exon Intron Exon Intron etc regulatory sites (cis)

Silent mutations with the same protein-AA sequence-Intronic Interchange of similar amino acids (hydrophobic or polar) Substitution of polar to nonpolar amino acids; changes in protein structure active site, regulatory regions resulting in from none to various levels of function

Genotyping

* The measurement of genetic variation ; SNPs are one of the most common types of genetic variation and can be used as markers of genes/polygene loci.

*Use of SNPs: genome wide marker coverage and allele discovery. There are 7 M validated SNP s in the human genome ; SNPs have also been widely identified in rodents and farm species

*SNPs may be located within a gene (promoter, coding or non-coding regions) or in the gene’s proximity. The marker (no-effect on transcription or non-synomynous ) type SNPs are easier to identify than those SNPs (synomynous) which in fact can affect transcription-splicing sites attributes. Many of the trait related SNPs are found in non-coding regions..

Kits, Kits everywhere

• There are proprietary reagents/kits available for nearly all applications in genomics and transcriptomics.

• Compare that to the Sambrook et al “Cloning Manuals” where one made all reagents.

• We rely on kits, but sometimes to our peril!

• Kits however have also opened many new avenues for molecular/genomics research at a reasonable cost for many that did not have in depth training in these techniques, but be cautious and beware of pitfalls of “kitology”.

• Workers may also use “service centers” to obtain genomic

data sets from their experimental samples.