Targeted Genome-Wide Enrichment of Functional Regions

Only a small fraction of large genomes such as that of the human contains the functional regions such as the exons, promoters, and polyA sites. A platform technique for selective enrichment of functional genomic regions will enable several next-generation sequencing applications that include the discovery of causal mutations for disease and drug response. Here, we describe a powerful platform technique, termed “functional genomic fingerprinting” (FGF), for the multiplexed genomewide isolation and analysis of targeted regions such as the exome, promoterome, or exon splice enhancers. The technique employs a fixed part of a uniquely designed Fixed-Randomized primer, while the randomized part contains all the possible sequence permutations. The Fixed-Randomized primers bind with full sequence complementarity at multiple sites where the fixed sequence (such as the splice signals) occurs within the genome, and multiplex amplify many regions bounded by the fixed sequences (e.g., exons). Notably, validation of this technique using cardiac myosin binding protein-C (MYBPC3) gene as an example strongly supports the application and efficacy of this method. Further, assisted by genomewide computational analyses of such sequences, the FGF technique may provide a unique platform for high-throughput sample production and analysis of targeted genomic regions by the next-generation sequencing techniques, with powerful applications in discovering disease and drug response genes

IS6110 FAFLP PCR, a tool for genomic mapping enabling investigation of evolutionary relationships of Mycobacterium tuberculosis in resource poor settings

Tuberculosis (TB) is an important communicable disease affecting the human population world-wide. Despite the efforts of the scientific community, national governments and WHO in controlling the disease, TB still remains a major killer in resource poor settings. New rapid assays and techniques that are simple and cost-effective are urgently needed to identify, treat and understand pathogenesis including the geographical distribution of the disease. The aim of the thesis is to develop a novel genomic mapping tool using Insertion Element, IS6110 that could aid in epidemiological studies of Mycobacterium tuberculosis complex (MTBC) in low and middle income countries. IS6110, a bacterial transposon, plays an essential role in changing the physical and biochemical traits of MTBC. Due to their transposition in TB genomes, they are used as epidemiological markers for differentiation of TB organisms and the mapping of these elements could also shed light on the putative altered function of adjacent genes. In the era of Whole Genome Sequencing (WGS) where repeat elements are difficult to sequence with short read technologies, a rapid and simple method of insertion site mapping using IS6110 FAFLP PCR was developed. This work is aimed at developing a rapid, cost-effective and robust genomic tool box exploiting the IS6110 FAFLP PCR assay that can both identify and characterise the TB genotypes / genetic lineages in any geographical location. For the first time using the assay above, TB samples from Nepal were categorised into different genetic lineages. Fifty-five percent of the samples analysed belong to Principal Genetic Group 1 (PGG1), Beijing and Central Asian strains. Also, new primers were designed targeting the Beijing and the T- groups using the FAFLP derived data that gave rise to the development of rapid lineage specific PCR assays. In addition, it was noticed that 3.9% of the Nepalese strains tested in this research work were likely multi-drug resistant (MDR-TB) using PCR targeting the Rifampicin-resistance-determining region (RRDR) of the rpoB region. It is demonstrated here that IS6110 FAFLP methodology could easily characterise the TB samples into different genetic lineages provided they have more than four IS6110 copies. In addition, lineage specific PCR does not need any expensive instruments or reagents except for PCR blocks and gel visualisers, and could be very effective in the rapid identification of different TB genotypes within hours. These data also add to knowledge about the circulating strains of TB in Nepal, currently a poorly characterised region of the world in this regard, and could help in contact tracing studies by epidemiologists. The IS6110 FAFLP technique thus can be employed in any geographical location to map TB genetic lineages where there is little or no information available on the prevailing TB strains

Synthetic Biology

Synthetic biology gives us a new hope because it combines various disciplines, such as genetics, chemistry, biology, molecular sciences, and other disciplines, and gives rise to a novel interdisciplinary science. We can foresee the creation of the new world of vegetation, animals, and humans with the interdisciplinary system of biological sciences. These articles are contributed by renowned experts in their fields. The field of synthetic biology is growing exponentially and opening up new avenues in multidisciplinary approaches by bringing together theoretical and applied aspects of science

Diagnostics in Plant Breeding

“Diagnostics in Plant Breeding” is systematically organizing cutting-edge research reviews on the development and application of molecular tools for the prediction of plant performance. Given its significance for mankind and the available research resources, medical sciences are leading the area of molecular diagnostics, where DNA-based risk assessments for various diseases and biomarkers to determine their onset become increasingly available. So far, most research in plant genomics has been directed towards understanding the molecular basis of biological processes or phenotypic traits. From a plant breeding perspective, however, the main interest is in predicting optimal genotypes based on molecular information for more time- and cost-efficient breeding schemes. It is anticipated that progress in plant genomics and in particular sequence technology made recently will shift the focus from “explanatory” to “predictive” in crop science. This book assembles chapters on all areas relevant to development and application of predictive molecular tools in plant breeding by leading authorties in the respective areas

Study of the vaginal and rectal microflora in pregnant women, with emphasis on Group B streptococci

In a first part of the PhD research we established the degree of correspondence between the vaginal and the rectal microflora. The composition of the human vaginal microflora is affected by several host factors, including, among others, age, menarche, hormonal changes, sexual activity, pregnancy and the use of contraceptives or spermicides, as well as individual habits such as douching (405). Studies of vaginal lactobacilli have demonstrated that Lactobacillus crispatus, L. jensenii, L. gasseri and L. vaginalis are the most commonly recovered species of H2O2-producing lactobacilli (5, 172, 287, 380) and the absence of H2O2-producing lactobacilli from the vagina has been associated with an increased risk for bacterial vaginosis (BV) (161, 241). BV is a condition whereby the lactobacilli are overgrown by anaerobic bacteria, such as Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Mycoplasma spp., Peptostreptococcus spp. and Prevotella spp. BV has been linked to increased shedding of HIV in the female genital tract (63), increased acquisition of HIV (161) and herpes simplex virus type 2 (63, 161) and with preterm birth (174). Several bacterial species are known to colonize both the gastrointestinal and the reproductive tract, and the rectum may play an important role as a source or reservoir for organisms that colonize the vagina (6, 238). The question remains whether these vaginal organisms are endogeneous or originate from the gastrointestinal tract. To establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimen from pregnant women at 35-37 weeks of gestation as part of the group B streptococci (GBS) screening program to prevent GBS neonatal disease, we identified the isolates to the species level with tRNA intergenic length polymorphism analysis (tDNA-PCR) and genotyped the isolates for those subjects from which the same species was isolated simultaneously vaginally and rectally, by RAPD-analysis (100). Also we compared the genotype of GBS isolates between the vagina and rectum (99). To further establish the potential role of the rectum as a reservoir for the vaginal microflora, we quantified the bacterial loads of six of the vaginally most important species by quantitative real-time PCR (qPCR), for those women where these species were present simultaneously in both niches (96). In summary, the species composition and the genotype similarity for those species simultaneously present in both niches, were unexpectedly high, indicating that indeed the rectum is probably the most important source of the vaginal microflora, or that strong dynamic exchange between both niches exist. This was further confirmed by the finding that for five of the six species tested (except for G. vaginalis), the vaginal and rectal bacterial load were in strong correspondence. A second part of this PhD research regarded improving the prepartum detection of group B streptococci. Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Early neonatal GBS infection can be prevented by identifying high-risk pregnancies and administering intrapartum antibiotics. Two different strategies, the screening-based and the risk-based approach, are used today in order to decrease the incidence of early-onset neonatal infection. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim Broth (Todd–Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar (57). However, this procedure may require 48 h to complete and its sensitivity might be improved. We compared different sampling and culture techniques for the detection of group B streptococci (98) and found that rectovaginal sampling was the most sensitive method for sampling and that Granada agar and Chromagar were the most sensitive culture techniques, only slightly improved by Lim broth enrichment. Also we compared two different targets (sip and cfb genes), using two different real-time PCR (qPCR) approaches, i.e. the hydrolysis probe (Taqman, Roche) and hybridisation probe (Hybprobe, Roche) techniques, directly on the clinical sample and after Lim broth enrichment, with the previously optimized culture approach (97), i.e. Lim broth enrichment, followed by subculture on Chromagar (97). We could establish that the sensitivity of these molecular techniques is significantly higher than that of the CDC recommended culture approach and also than that of our improved culture approach. Rather surprisingly, the sensitivity of these molecular techniques is further significantly improved by Lim broth enrichment compared to direct application on the clinical sample. The consequences of this observation for the application of the rapid combined DNA extraction/qPCR techniques, as they are now available for intrapartum screening, are still unclear, and will be the basis for future research in the rapidly evolving GBS diagnostics

Nucleic acid based molecular biological methods for quantitation and characterization of human cytomegalovirus in the clinical specimens from immunocompromised patients

The main objective of the study was to develop and establish the usefulness of nucleic acid based molecular methods for detection and quantitation of HCMV in various clinical specimens from patients with clinically suspected HCMV infections. In addition the HCMV strains encountered in these patients were characterized based on five different glycoprotein genes viz. glycoprotein B belonging to gCI complex; glycoprotein H, glycoprotein O and glycoprotein L belonging to gCIII complex and glycoprotein N belonging to gCII complex. ♦ pp65 antigenemia assay, a rapid, sensitive and semi-quantitative assay based on the detection and quantitation of lower matrix protein pp65 of HCMV for detection of active HCMV infection was standardized. Initially, two different methods namely the Conventional Dextran sedimentation (CDS) and Direct erythrocyte lysis (DEL) were compared for isolation of leucocyte from peripheral blood. Since, a good agreement in the quantitation of pp65 positive cells was found between the two methods, they were alternatively used in the rest of the study. pp65 antigen was not detectable in the peripheral blood of the controls (healthy blood donors seropositive for HCMV) confirming the earlier view that detection of the antigen in a patient is an evidence for active HCMV infection. ♦ Since, nucleic acid based molecular methods showed great promise in detection of several viruses and also reports suggesting that the region of interest can affect the sensitivity of the Polymerase chain reaction, three different targets viz. mtrII, UL-83 and gO gene of HCMV were evaluated against pp65 antigenemia assay as ‘gold standard’. PCR for mtr II was found to be more suitable for qualitative detection of HCMV in Chennai region, India. The other findings from the study include the inability of the qualitative PCRs to detect latent viruses circulating in the seropositive controls and all the three regions becoming positive with high level of antigenemia (>50 cells/ 2x 105 PBLs). ♦ Since, Post renal transplant recipients and HIV patients with HCMV infections remain asymptomatic with lower viral load, quantitation of HCMV in these patient groups become increasingly important. Also, since the results of the previous study suggested a high viral load when all the three regions became positive, multiplexing of the three primers were attempted. The multiplex PCR for the three regions of HCMV proved to be useful in quantitation of HCMV since it generated four different patterns that segregated with different pp65 antigenemia levels. ♦ Taqman probe based Real-time PCR was standardized for the mtrII region of HCMV. The mtr II region was chosen as it provided 100% sensitivity for detection of HCMV DNA in immunocompromised patients. The real-time PCR was sensitive, rapid (results available in 2 hours) and had a high reproducibility. It had an ability to detect the HCMV genome circulating in peripheral blood specimens of healthy controls. The median viral load in the healthy controls was 26.5 copies/ml. ♦ The multiplex PCR was evaluated against pp65 antigenemia assay and Taqman-probe based real time PCR for its efficacy in quantitation of HCMV. Each pattern of the multiplex PCR was assigned a viral load based on the results of Taqman-probe based real time PCR. The median antigenemia levels and viral loads in clinical specimens showing Pattern I and Pattern II of the multiplex PCR was significantly higher than Pattern III and Pattern IV. A moderate positive correlation ((rs) = 0. 7778) was seen between the results of pp65 antigenemia assay and Taqman – probe based real time PCR in the study. The multiplex PCR was less expensive and had the ability to distinguish clinical specimens with high and low viral load which may be effective in predicting patients who might develop HCMV disease and to monitor effectiveness of antiviral therapy. ♦ Since, the genotypic difference of HCMV affects pathogenesis and tropism of HCMV, the genotypic methods for determining the distribution of HCMV genotypes in the study population was developed and evaluated. ♦ Initially, a nested PCR based RFLP for gB gene was attempted however the method was unable to identify genotype gB3 in the study population. The study indicated that the sequence variations in the gB genome of HCMV may complicate genotyping of HCMV. Confirmation of primer specificity by the BLAST program may not suffice for the genotyping studies. Complete alignment of the prototype sequences of all the genotypes that exist for the gene of interest by a multiple alignment program with the primers used for amplification may be necessary for genotyping of HCMV. ♦ Later, seminested PCR-based RFLP and multiplex PCR were developed for genotyping of gB gene. Multiplex nested PCR for gB gene was more advantageous than the commonly employed PCR based RFLP for gB genotyping since it was rapid and allowed easier detection of mixed infections with multiple gB genotypes in clinical specimens. ♦ In the study determining the distribution of gB genotypes by multiplex PCR a predominance of gB1 genotype followed by gB3 and gB2 irrespective of the patient group or specimen type was found. Absence of gB4 and gB5 in the study population and gB mix in the infant group was observed. Presence of gB2 in the intraocular fluid was statistically significant. No absolute segregation of gB genotype based on gender or patient group was observed. In the HIV patient group with discordant results between the blood and intraocular fluids, the genotype other than gB1 in the blood, was detected in the intraocular fluid. ♦ PCR-based RFLPs were standardized to determine the distribution of genotypes pertaining to the genes of gCIII complex viz. gH, gL and gO. In all the three cases, the existing protocols were modified to achieve the following: direct application on clinical specimen without requirement for isolation, detection of mixed genotypes and visualization of restriction products on agarose gel. ♦ The analysis revealed no significant differences in the distribution of the gCIII variants based on gender. Statistically significant difference in the distribution of gH genotypes and gO genotypes were found in different patient groups. gL1 and gL2 genotypes were not found in the study population and predominance of gL4 genotype irrespective of the patient groups and specimen types were also observed. Fifty percent of HIV patients in the study was shown to harbour HCMV strains with gH1-gO1-gL4 configuration. ♦ An existing uniplex PCR based RFLP was converted to seminested PCR based RFLP to attain optimal sensitivity for genotyping gN gene of HCMV strains from direct clinical specimens without the need for viral isolation. The distribution of gN genotypes in the study population was in accordance to the distribution seen in other geographical areas. There was no statistically significant difference in the distribution of gN genotypes with respect to gender or clinical specimens. The genotype gN4c predominated among the genotypes irrespective of the patient types or specimen though its frequency was little less in PRT than other groups of patients. ♦ Following conclusions were drawn from the studies on cumulative genotyping of the five regions of HCMV: The study confirms that HCMV are highly heterogenous group of viruses and exhibit several genetic combinations. Theoretically 1120 strains may exist based on the assumption of number of genotypes existing for the five glycoprotein genes and the present study showed 48 different strains circulating in the study population. Genetic linkages are limited though some associations of genotypes occur more frequently than others. Hence, variation in one part of the gene does not reflect variations in other parts of HCMV genome. Analysis of single genes may not aid in understanding the tissue tropism or pathogenesis of HCMV. Infection with one HCMV strain does not provide protection against reinfection with a new strain, since a higher number of mixed infections with multiple genotypes were encountered in Post-renal transplant patients and HIV infected individuals. The order of genetic loci suitable for detection of mixed genotype as confirmed by the study is gB>gO>gH=gL>gN. The leucocyte fraction of peripheral blood is recommended for detection of mixed genotypes in PRT and HIV patients. A statistically significant difference was observed in the viral loads as determined by Taqman probe-based real time PCR between the specimens carrying single genotype and multiple genotypes. Hence, detection of mixed genotypes in the hypervariable regions of HCMV genome can be a surrogate marker for increase in viral load. The mixed genotype in any loci was not observed in the infant group in this study. ♦ Phylogenetic analysis of the four isolates and fifteen other HCMV strains revealed that no novel variant existed with respect to the regions of the gene analyzed and all the strains could be accommodated into one or the other genotypes already established