The whole genome sequencing to identify Mendelian disorders

The current way to determine the cause of disease is finding mutations via DNA sequencing. In order to reduce costs only coding regions are sequenced and analyzed. Unfortunately, several Mendelian traits that can be the basis for specific diseases are not present in coding regions. Therefore, the sequencing of the whole genome might contribute better understand the causal variant of diseases. Scientists from the Institute for Systems Biology in Seattle proposed this approach to study the Mendelian hesitance of two recessive disorders. They analyzed the whole genomes of healthy parents and sick children. They delineated an accurate recombination map showing exactly which pieces of parental chromosomes had been assembled in offspring genetic material. Then, they corrected 70% of sequencing errors and especially they reduced the search space for the disease- causing variants. This study is important because it demonstrated that is possible to identify the genes involved in etiology of certain disease by sequencing the DNA of the family in which this disease appears. Based on this observation, scientists plan to analyze the genome of family with Huntington’s disease. This approach requires the absolute precision of sequencing data.

Replication timing analysis

The replication timing assay is a current lab method used to determine the exact phases of replication process. Exponentially growing cells are labelled with 5- bromo 2-deoxyuridine to mark newly generated cells. Indeed, the bromo-deoxyuridine is incorporated into DNA in place of thymidine. Fluorescent dye DAPI stains the whole cellular DNA and enables to fractionate cells on the basis of total amount of DNA and thus, the cells’ stage of the synthesis phase of cell division.
Then, labelled DNA is isolated by immunoprecipitation and analysed by PCR on the loci of interest. A recent paper published this year in PNAS, proposes to substitute the PCR with the DNA assembling into separate Illumina sequencing libraries and sequence it. It seems that the most of the genome is going to be replicated at about the same time in different cell types, but the study of other cells populations will be clarified the timing. Furthermore, sequencing will permit analysis also of rare cell populations because of the low number of cells required for the assay. In conclusion, this new approach of replication timing assay allows extending many analyses at the whole genome levels, improving the throughput and accelerating scientific advancement.

New technologies to identify gene function

After advances in DNA sequencing technology, the major task is to determine the functional role of proteins coded by these sequenced genes. Given the broad range of different functions carried out by proteins, it’s obvious that a multiplicity of techniques will be necessary, while DNA sequencing is achieved by few, easy and simply technique. A series of strategies based on generalization and systemization of genetics are emerging now as important tool to fill the gap between sequence and activity. One of these approaches is the analysis of the effect of perturbations of gene expression, by deletion, mutation or over-expression: after one of these modifications, we expect to observe a phenotypic change.

The challenge is to quantitatively measure phenotypes with enough accuracy and depth to define gene function. Two complementary approaches for determining complex phenotypes are currently used: in the first one many different parameters are simultaneously analysed, this is an high content screen; otherwise a single or limited number of aspects are observed, but the effect of perturbing each gene is followed in combination with a second perturbation, either another mutation or a chemical treatment. This genetic interaction profiling offers a high-resolution view of the function of each gene. Saccaromyces Cerevisiae is a model really useful for this kind of studies: a complete series of deletion strains of nonessential genes has been produced and has allowed to better understand the role of proteins important for yeast biochemistry and biology. Important results have been achieved also by using conditioning mutants that selectively grow in rich media: also in this case precious information has been retrieved. Rather than loss of function studies, methods for systematic gene over-expression have been optimized. Novel approaches in this field are interested in construction of untagged proteins in order to exclude that the presence of tag could interfere with the normal function of protein self.

The main goal of this systematic studies is to maximize the information flow, while minimally compromising the accuracy of phenotype detection. The introduction of large biomolecules into cells, such as DNA, RNA allows to directly analyse the role of one gene in the cellular life, and different kind of cells (mammals, primary cells, stem cells) can be used in this approach. Biomolecule is printed in an array onto glass slides, as done in conventional microarray. A monolayer of cells is deposited on top of the arrayed molecules and cells are transfected by taking up the material from glass. By using 96-well format plate, it’s possible to analyse the effects of a large number of biomolecules in a quantitative way. A plausible example of this method application is the effect of iRNAs on cellular proliferation: iRNA can be printed on bottom plate, cells are transfected (please note that is important to define the efficiency of transfection) and proliferation rate can be measured with normal treatment with MTT. In this way, genes important in proliferation could be identified.
Future efforts will be done to exploit a vast array of data that will emerge from large-scale genomic and proteomic projects to gain a deeper knowledge of the function of biological system.

Manipulation of bacterial genome in yeast

Even if manipulation of bacterial genome is often difficult and challenging, engineering allows to better understand bacterial biology and genetics. Researchers from C. Venter Institute improve a protocol to clone bacterial genome in yeast, manipulate it and boot it up in bacteria self. To do this they chose an “easy” model, Mycoplasma, because this organism doesn’t have bacterial wall, its genome is small and A-T rich, so is more properly replicated in yeast than ones rich in G-C. Furthermore Mycoplasma has non-standard genetic code that can not be translated in yeast, preventing the synthesis of bacterial proteins toxic for yeast.

What did scientists perform to achieve this important result? They cloned Mycoplasma genome into yeast artificial chromosomes (YACs), genetically manipulated it and then transplanted it into the final organism receiver. Two concerns could prevent this goal: one was the possibility that restriction endonucleases recognised foreign sequences and degraded them and the second one was that yeast modified bacterial genome. Fortunately this last event didn’t occur, while to limit endonucleasic activity, scientists hypermethylated donor genome and eliminated endonucleases from receiver organism. This protocol could be improved in order to become a conventional technique for bacterial manipulation in order to have another tool to solve human needs in medicine and environmental preservation.

Direct RNA sequencing

Recent paper published on Nature describes an innovative method to directly sequence RNA. mRNA is usually isolated from total cellular lysate and converted in cDNA by reverse transcriptase; then, cDNA is sequenced with protocols currently used for DNA sequencing. The use of an enzyme for retro-transcription could insert mistakes into cDNA sequence with quite high frequency: an advantage of direct RNA sequencing is to bypass this step.

How does RNA sequencing occur? Polyadenylated RNAs are bound onto solid support; DNA polymerase able to use RNA as a template binds one fluorescent nucleotide complementary to the first basis of RNA. This binding generates fluorescent signal, that is read. After reading, fluorescent probe is cleaved and the following basis on RNA can be recognized and bound to another fluorescent nucleotide. In this way, after a fixed number of cycle, all RNA molecule is sequenced. Even if this technique is innovative and extremely useful, it needs to be further improved and tested because some problems due to RNA stability could arise. Moreover, it has to be ascertained the proficiency of DNA polymerase that must perfectly work. However, we hope that this method could enter as soon as possible in clinical practice.

High-throughput re-sequencing to identify rare allelic variants

Recent advances on genomic studies allow to re-sequence genes with high accuracy and in high throughput procedure. Rare allelic variants are important to be analyzed because are often the molecular basis of human disease. Indeed, numerous syndromes are associated to mutations in rare allele, but a lot of work have to be done yet.

A new protocol has been published to apply an array to high throughput re-sequencing. Two problems were encountered at the beginning of the study: the upstream target preparation techniques available until now were not able to produce thousands of samples simultaneously and the accuracy was too low to distinguish rare variant to false positives. Scientists from Genentech and Stanford Genome Technology Center developed a method for target amplification by capture and ligation (TACL) based of novel probes for genomic DNA that are amplified by PCR, incorporate deoxyuridine and are purified. TACL method provided high reproducibility ad specificity in terms of capturing of the target regions also when the starting sample concentration was lower than 15 nanograms. Then, they cloned TACL probes into bacteria and used them to hybridize to probes previously captured. The bacterial growth in selective media allowed to recognize where the mismatched was preset, thus enriching the rare alleles. This technique seems promising and user-friendly because doesn’t need of particular instrumentation to be successfully employed.

New method to quantify rare single-nucleotide polymorphism

Recent studies correlate rare allelic variants to many complex traits and combined effects of this deleterious mutations could explain susceptibility to many common diseases. To identify rare variants it’s necessary to genotype high number of individuals, by sequencing, or a pooled sample to minimize costs.

Druley and co-workers proposed a combination of molecular biology and computational analysis to achieve targeted resequencing and rare-variant detection. Procedure required PCR-amplification and sequencing with Illumina. They found that the first 12 bases of each Illumina read contained significantly fewer errors than later and, so, they used only this portion for their analysis.
They developed a new algorithm based on large deviation theory, named SNPSeeker. This program uses a second-order dependency error model for single-nucleotide polymorphism identification and considers the position of sequencing read (PCR cycle number) and the identity of two upstream bases. Thus, they improved the specificity of SNP calling and obtained results comparable to Sanger sequencing data, consistently reducing costs.
An important application of this method, is the combination of pooled-sample sequencing with genomic selection strategy to perform a more systematic survey of protein-coding DNA. This knowledge would be an important achievement for disease screening and tailoring risk-appropriate therapy.