NEXT GENERATION SEQUENCING

Genome Enrichment Questions: Conventional PCR is not effective in preparing the starting material for NGS. A machine is powerful but we are unable to give them enough input. How do you enrich the genome? How do you increase the number of input into the machine? Answer: a) Use micro-droplet PCR technology Simultaneous amplification of 3,976 products o A microfluidic device creates aqueous picolitre-volume droplets of forward- and reverse-targeting primers in an oil solution. o The primer droplets are targeted to different regions of the genome. o They merge with separate picolitre droplets that contain fragmented genomic DNA and associated PCR reagents, and these mixed droplets are thermal cycled in a single tube The authors reported an 84% capture efficiency with 90% of the targeted bases showing uniform coverage using the micro-droplet PCR method

b) Solid Phase Custom-designed oligonucleotide microarrays and solution-based hybridization strategies have also been used for targeting regions of interest. o Roche/NimbleGen oligonucleotide microarrays: designed to provide complementary sequences for solid phase hybridization to enrich for exons or contiguous stretches of genomic regions. o The BCM-HGSC and Cold Spring Harbor Laboratory groups reported capture efficiencies of: 6577% Roche/454 platform 53%(Illumina/Solexa platform, with targeted exons being covered by at least one NGS read. After NGS reads have been generated, they must either be aligned to; Known reference sequence or assembled de novo. De novo assemblies o reported for bacterial genomes and mammalian bacterial artificial chromosomes o substantial challenges exist for their application to human genomes When do we need to use these methods? The decision to use either strategy is based on the intended biological application as well as cost, effort and time considerations. For example; o You want to identify & catalogue genetic variation in multiple strains of highly related genomes; o specific species of bacteria, C. elegans or A.thaliana, o Align NGS reads to their reference genome is substantially cheaper and faster than Sanger sequencing. Single Nucleotide Variants can be readily identified, although in many cases, validation of these findings is required. There are limitations to the alignment approach; o Aligning reads in repetitive regions in the reference genome or in regions that may not exist in the reference genome; The latter as a result of gaps in the reference genome or the presence of structural variants (SVs) in the genome being analysed. Structural variants: All sequence variants other than single-nucleotide variants, including;

Block substitutions, Insertions, Deletions, Inversions, Segmental duplications, Copy-numberdifferences.

Alternative to conventional sequencing semi-conductor (SC) to get the sequence of reads how SC function? How it can used to replace conventional imaging? Name of the method? Describe, how it function? Used protons instead of Sanger method, explain how the protons changes the pH level indicates the addition of new nucleotide. Principle?

Answer: CMOS A. Simplified drawing of a well, a bead containing DNA template, and the underlying sensor and electronics. A. Protons (H+) are released when nucleotides (dNTP) are incorporated on the growing DNA strands, changing the pH of the well (pH). B. This induces a change in surface potential of the metal-oxide-sensing layer, and a change in potential (V) of the source terminal of the underlying field -effect transistor. B. Electron micrograph showing alignment of the wells over the ISFET metal sensor plate and the underlying electronic layers. C. Sensors are arranged in a two-dimensional array. A. A row select register enables one row of sensors at a time, causing each sensor to drive its source voltage onto a column. B. A column select register selects one of the columns for output to external electronics. The Ion Torrent PGM (Personal Genome Machine) from life technologies; Uses CMOS integrated circuits to sequence. It has micro-machined reaction wells, each of which holds a different template. Beneath the wells is an ion-sensitive layer and beneath that a proprietary Ion sensor.

Transgenic Technology 1. Transgenic animal A) DNA microinjection: Direct microinjection of a chosen gene construct into the pronucleus of a fertilized ovum

o A single gene or a combination of genes from another member of the same species or from a different species Introduced DNA may lead to; o Over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The insertion of DNA is, random and there is a high probability the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilised ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male. o Advantage: Applicable to a wide variety of species.

B) Embryonic stem cell-mediated gene transfer. Insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. o These cells are then incorporated into an embryo at the blastocyst stage of development. o The result is a chimeric animal. o ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method. This technique is of particular importance for the study of the genetic control of developmental processes. It works particularly well in mice & has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination. C) Retrovirus-mediated gene transfer. Gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.

2. Transgenic Plants a) Agrobacterium mediated; Ti-plasmid based expression construct is made and introduced into agrobacterium. Leaf of a plant is scratched and dipped into solution of transformed agrobacterium resulting in the infection of the plant by the bacterium. The Ti-plasmid construct is transferred into the plant

genome and callus forms. Grow callus on selective medium (with phytohormones) to develop root and shoot system to regenerate transgenic plant. Advantage: close to natural infection and callus formation. Integration is more certain, greater frequency of single-site insertions of the foreign DNA making it easier to monitor, the process is more controlled and is reproducible. Limitations: a) Not all plants are as susceptible to infection by agrobacterium b) More challenging for monocots (eg. cereal crops) Perhaps because wounding of monocots does not produce appropriate types/ amounts of phenolics? o vir genes not expressed perhaps because monocots respond differently to wounding o wounding activates cell division in dicots o wounds become lignified in monocots b) Gene gun - Microprojectile bombardment (biolistics) Tiny gold or tungsten particles are coated with the recombinant construct and fired into the plant tissue (leaf, callus, cell culture). Some lands in the cell nucleus and can be incorporated into the genome. outcome is variable need to optimise particle size particle acceleration amount of DNA has been successful for a range of species cereal crops sugar cane

Advantage: can be used for monocots which do not easily infect with agrobacterium Limitation: cannot control integration into the host genome. Integration is random outcome C) Electroporation/ transformation of protoplasts Protoplasts are mixed with DNA construct

o eg. plasmid containing desired transgene Mixture exposed to brief electrical pulse (high voltage) Foreign DNA enters the host cell Direct introduction of DNA into monocots by electroporation has been successful Advantages: can be used for monocots which do not easily infect with agrobacterium Limitation: electroporation technique is rarely used cf. other methods difficult to regenerate entire plants from protoplasts especially for woody plants

What is chimeric animal what is transgenic animal? Explain the diff. How the diff arises? Where is it come from? Transgenic mice are created by microinjecting DNA into already fertilized mouse eggs. Chimeric mice are made by microinjecting embryonic stem (ES) cells into mouse blastocysts.

Advanced PCR Advanced electrophoresis method 1) DGGE, TGGE & SSCP All based upon the fact that double stranded DNA will melt at a specific concentration of denaturant or at a specific temperature The melt profile is sequence dependent When dsDNA melts its mobility in a gel is reduced then stops 2) Single strand conformation polymorphism (SSCP) is: Conformational difference of single stranded nucleotide sequences of identical length. Different conformations can be separated by gel electrophoresis. 3) Denaturing Gradient Gel Electrophoresis (DGGE)

 Increasing gradient of denaturant through gel; dsDNA melts as it reaches a particular area where denaturant concentration is high enough. From low to high concentration (dsDNA partial ss ssDNA)

4) Thermal Gradient Gel Electrophoresis (TGGE) Similar to DGGE, except that the dsDNA is melted by increasing temperature of gel Generate samples for analysis by PCR with GC-clamped primer adaptors - Strands cannot then come apart completely Separate into two: i. Perpendicular; - One sample separated over a broad temp range, temperature gradient is perpendicular to the movement of the sample through the gel. Used to find the optimal melt temperature for separation. ii. Parallel; - Multiple samples are analysed in a temp gradient parallel to the direction of migration. Used more for the routine analysis of samples. 5) Constant Gradient Gel Electrophoresis Modification of DGGE; using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments. This leads to increased resolution of mutants as fragments differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains.

Gene expression 1. How do we target mRNA? a. Give your opinion, how to prevent the expression of the gene? b. Antisense RNA @ Ribozyme? Either one only c. Describe, principle, how do it helps to control the expression of the mRNA. d. Provide examples 2. Need to explain the application of this technology Antisense RNA Antisense RNA has sequence that is complementary to mRNA. It can therefore form duplexes with mRNA and may interfere with gene expression.

Stable production of antisense RNA has been shown to inhibit mRNA processing and to prevent translation - may interfere with mRNA processing - may target mRNA for degradation - may prevent ribosome attachment

Whereas many traditional drugs block the activity of disease causing proteins, antisense technology may be used to prevent the production of harmful proteins.

Application I. The FLAVR SAVR tomato first genetically modified food approved by the FDA (1994) poses no risk to either the environment or consumers change was not great enough to mandate GM labelling insertion of an additional copy of the polygalacturonase (PG) gene in the "antisense" orientation reduces translation of endogenous PG mRNA PG enzyme degrades pectin leading to fruit softening FLAVR SAVR tomato ripens normally, but less pectin breakdown stays firm after harvest, so can be left on the vine longer easier to handle during packaging and transport

created by Agrobacterium-mediated transformation T-DNA contained tomato PG gene in the antisense orientation T-DNA also encoded selectable marker (kanR) -non-toxic protein, inactivated/ degraded in the gut no incorporation of plasmid DNA sequences outside of the T-DNA region hybridization of antisense and sense mRNA transcripts reduces mRNA available for protein translation PG activity in FLAVR SAVR tomato < 1% of PG activity in parental line no translation of the antisense-PG gene - no novel proteins produced nutritional value of tomato unaffected no changes in levels of potential toxins (natural glycoalkaloids) disease, pest and horticultural traits shown to be unchanged OR Gene therapy for hepatocellular carcinoma (HCC) (therapeutic)

II.

HCC is one of the most common malignancies; the prognosis of this cancer is very poor. Efficient tumour cell invasiveness and secondary tumour formation (metastasis) are associated with the release and activation of proteinases in the peritumoral microenvironment by tumour and/or stromal cells. Clinical findings and experimental evidence strongly indicate that urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR) are linked to the degradation and remodelling of normal and cancer tissue and of the surrounding extracellular matrix. In particular: u-PAR gene expression is upregulated in HCC u-PA gene transcription is activated in HCC levels of u-PA mRNA expression are inversely correlated with patient survival Therefore, the plasminogen activation system (u-PA/u-PAR) is considered a potential target for cancer therapy by antibodies, enzyme inhibitors, synthetic u-PA and u-PAR analogues, antisense oligonucleotides and antisense u-PA. Researcher showed that stable expression of antisense urokinase mRNA inhibits the proliferation and invasion of human hepatocellular carcinoma cells, as shown overleaf. Their work provides evidence that u-PA antisense technology might be an efficient strategy that could provide an improved prognosis for HCC sufferers.

Ribozyme Ribozymes are catalytic RNA molecules that specifically cleave target RNAs. Ribozymes targeted to mRNA molecules can effectively prevent translation and prevent gene expression. A single ribozyme can cleave many target mRNAs, which provides a significant advantage over antisense technology (one antisense-RNA binds to one mRNA molecule, so high levels of antisense expression are required).

Application Ribozyme use in Cancer Treatment- Researcher developed a specific trans-splicing ribozyme that targets telomerase (hTERT) RNA in human telomerase-positive cancer cells. The ribozyme recognizes hTERT RNA by base pairing to the internal guide sequence of the telomerase enzyme. The ribozyme then removes the sequence downstream of the target site and replaces it with a 3' exon that encodes a cytotoxin RNA sequence. Expression of the therapeutic cytotoxin gene occurs selectively in cancer cells that express the hTERT gene, leading to reduced survival of cancer cells and a reduction in tumour size.

GENE Therapy

A-GENE AUGMENTATION THERAPY(exchange non-functional gene to functional gene) -Treatment of inherited disorders caused by the loss of a functional gene product. -Functional copy of absent gene provided, must be expressed: *in the appropriate tissue *at the appropriate time *in sufficient quantity -Only effective if the pathogenic effects of the disease are reversible. B-GENE SUPPRESSION THERAPY (gain inappropriate gene which we dont want it to be expressed) -Treatment of infectious diseases, cancer and inherited disorders caused by inappropriate gene activity -Aim to introduce a gene whose product inhibits the expression of the pathogenic gene and/ or interferes with the activity of its product *may interact with gene directly *may interact with control elements RNAi as therapy Example of disease: Huntington's disease (HD) and many spinocerebellar ataxias (SCAs) -caused by expansion of polyglutamine tracts -mutation promotes misfolding of the disease protein -initiates a complicated, still poorly understood, molecular cascade leading to neuronal dysfunction and cell death Alzheimer's disease (AD)

-caused by mutations in genes encoding amyloid precursor protein or the presenilins Amyotrophic lateral sclerosis (ALS) -caused by mutations in the superoxide dismutase 1 [SOD1] gene Frontotemporal dementia -mutations in the gene encoding tau (microtubule-associated protei Method: RNAi is a mechanism of silencing gene expression (after the gene has been transcribed) due to the presence of antisense RNA which is complementary to a specific cellular mRNA. The complementarity causes the two molecules to bind to each other 1. Double-stranded RNA found in the cell recognized by Dicer (an endonuclease) that cuts it into pieces that are 21 to 23 nucleotides long. An antisense RNA fragment can then associate with a complex of proteins to form the RISC 2. The silencing complex then associate with cellular mRNA that has a region of sequence homology with the RNA fragment in the RISC 3. The mRNA is then degraded. The presence of this rapid degradation mechanism reduce and effectively eliminate translation of mRNA for the gene, thus interfering with gene expression.

or Discuss methods that might be used to achieve both stable and transient gene silencing. -Transient, introduce the synthetic siRNA to the host cells, will behave like an endogenous and engage with the RISC complex to bring about silencing.

-Stable; require the host cells to either carry an expression vector producing an siRNA or to have the construct (siRNA producing) incorporated into the host genome

Briefly detail an example of applications for which these techniques might be useful.

-Cancer, genetic and multifactorial disease, can use siRNA to identify causal factors and determine therapeutic potential

Molecular epidemiology Typing method : genotypic (3 types) SequenceTM 1-multilocus sequence typing (MLST) Application: -detects variation in the sequence of DNA fragment of 5-10 housekeeping gene *fragments typically 400-500bp * housekeeping genes essential to cell survival -PCR used to amplify the genes then product sequenced -each gene sequenced is considered an allele type *each unique sequence is designated a new number *an allelic profile forms the sequence type -dendogram can be constructed for isolated with similar allelic profiles where it can be assumed they share ancestry -MLST schemes available for >30 pathogenic bacteria and fungi (comparison of existing data reasonable straight forward) Advantage: -developed to assess interrelated for evolutionary studies -Good-inter laboratory comparison (library) -for L.pheunomphila, method has been developed to allow direct analysis of clinical sample

Disadvantage: -not well suited to discriminating for epidemiological investigation in localised area or within short time (eg: health centre acquired infection) -require a lot of PCR reaction and a lot of sequencing (Time and money) 2-single nucleotide polymorphism (SNP) Principle: -SNP detection ATTACGCGA
ATAACGCGA

-determine relationship among highly homogenous pathogens. Eg: M.tuberculosis, E.coli O157:H7, B.anthracis -based on MLST but applied to only the genes where variation common Advantage: -simple, cost-effective Disadvantage: -less discrimination and cannot detect new allelic type 3- single locus sequence typing Principle: -analysis of single variable genetic locus (opposed to multiple) -used for s.aureus on hypervariable region of spa gene and correlates very well with whole genome clonal variation (local and global application) Best used with the combination of other methods Advantage: -save workload, time and cost Disadvantage: Not widely used as lack of discriminatory power fragment TM 1-Restriction fragment length polymorphism (RFLP) Principle: -1st bact genotyping method -amplification of chromosomal DNA *standard PCR *product fragmented using selected restriction enzyme -R.E actives at restriction sites, 4-8nucleotides long, palindromic (rotor, kayak) Result: DNA fragment separated using gel electrophoresis -mutation results in loss/gain of restriction site.so different fragment profile Advantage: -can use more than 1 R.E (separately) Disadvantage:

-increase work but may lead to better differentiation -rarely used in modern molecular epidemiology 2-pulse field gel electrophoresis Principle: -uses macro restriction enzymes (8mers) -Whole bacterial genome separated into 10 30 fragments -Fragments may be ~1000 kb ( 1 Mb) or larger, thus impossible to separate on conventional gel (than 30 50 kb are large and will run at the same rate on conventional gel) -By changing direction of charge larger fragments realign slower than smaller fragments, so migrate slower: enables differentiation of large fragments Advantage: -PFGE versatile: has been used on large variety of bacterial pathogens -High discriminatory power -Particularly at the time of early application Disadvantage: -Gels take a long time to run: 12-48 hours -Time consuming and laborious - Reference protocols, databases etc established for some pathogens, BUT Inter-laboratory comparison difficult 3-multilocus variable no of tandem repeat Principle: - Appropriate regions of genome selected and primers that flank the region are used. Typically use published primers if they exist: allows comparison of data -Number of loci depends on evolution rate of pathogen (Typically 4 7, but can be more) -E.g. M. tuberculosis, ~ 20 loci -The more repeats, the bigger the product -Size determined by agarose elctrophoresis (rarely), capillary electrophoresis, mass spectrometry or sequencing -Variability of each loci should be assessed Advantage: -reasonably simple, rapid, inexpensive (Depending on number of loci) -In general, MLVA data correlates well with other genotyping methods