Algorithms for sequence-based reverse metabolic engineering

In bacterial genomes a fraction of transcribed sequences do not code for proteins or structural RNAs, but have been shown to be involved in fundamental processes, such as regulation of gene expression, mRNA processing and stability or structural RNA maturation. In this thesis a systematic procedure to identify and classify families of repeated sequences sharing a common RNA secondary structure was applied to the study of 40 bacterial genomes. Sequences able to fold in a stable stem loop structure were clustered according to sequence similarity, and grouped within homogeneous families. The study led to the identification of 57 families of repeated sequences, sharing a common secondary structure and potentially coding for structured RNAs. All previously known such families have been detected by the used procedure, and are listed within the final set, together with 37 novel ones. Their location in relation to protein coding genes was evaluated, and a correlation was found between structure and positioning within intergenic regions. A new software tool is also described, Scaffolder, designed to help in high-throughput de novo genome sequencing by finding connections between contigs produced by random shotgun sequencing, and assisting the researcher in the whole process. The software, accessible both as a command line tool and as a web application, can guide all the final phases of genome assembly by storing the current assembly status, displaying networks of connected contigs and untangling multiply connected ones by a combination of computational and experimental procedures.

With the development of new sequencing techniques, whole genomes of many species have become available. This huge amount of data gives rise to new opportunities and challenges. These new sequences provide valuable information on relationships among species, e.g. genome recombination and conservation. One of the principal ways to investigate such information is multiple sequence alignment (MSA). Currently, there is large amount of MSA data on the internet, such as the UCSC genome database, but how to effectively use this information to solve classical and new problems is still an area lacking of exploration. In this thesis, we explored how to use this information in four problems, i.e. sequence orthology search problem, multiple alignment improvement problem, short read mapping problem, and genome rearrangement inference problem. For the first problem, we developed a EM algorithm to iteratively align a query with a multiple alignment database with the information from a phylogeny relating the query species and the species in the multiple alignment. We also infer the query's location in the phylogeny. We showed that by doing alignment and phylogeny inference together, we can improve the accuracies for both problems. For the second problem, we developed an optimization algorithm to iteratively refine the multiple alignment quality. Experiment results showed our algorithm is very stable in term of resulting alignments. The results showed that our method is more accurate than existing methods, i.e. Mafft, Clustal-O, and Mavid, on test data from three sets of species from the UCSC genome database. For the third problem, we developed a model, PhyMap, to align a read to a multiple alignment allowing mismatches and indels. PhyMap computes local alignments of a query sequence against a fixed multiple-genome alignment of closely related species. PhyMap uses a known phylogenetic tree on the species in the multiple alignment to improve the quality of its computed alignments while also estimating the placement of the query on this tree. Both theoretical computation and experiment results show that our model can differentiate between orthologous and paralogous alignments better than other popular short read mapping tools (BWA, BOWTIE and BLAST). For the fourth problem, we gave a simple genome recombination model which can express insertions, deletions, inversions, translocations and inverted translocations on aligned genome segments. We also developed an MCMC algorithm to infer the order of the query segments. We proved that using any Euclidian metrics to measure distance between two sequence orders in the tree optimization goal function will lead to a degenerated solution where the inferred order will be the order of one of the leaf nodes. We also gave a graph-based formulation of the problem which can represent the probability distribution of the order of the query sequences.

Human tuberculosis disease (TB) is caused by bacteria within the Mycobacterium tuberculosis complex, including M. tuberculosis (Mtb). Genetic variation within the pathogen can lead to drug resistance, affect virulence and transmissibility. I have analysed Mtb whole genome sequence data to improve the understanding of global genetic variation, and the resulting insights could ultimately assist the development of TB control measures. Whole genome sequencing platforms are being used to infer drug resistance profiles, and thereby could assist clinical management. I investigated the reproducibility of sequence data from two platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (TBProfiler, Mykrobe predictor). DNA replicates from the reference strain (H37Rv) and 10 drug-resistant strains were sequenced, and inferred drug resistance genotypes were compared to drug susceptibility testing phenotypes. Genome-wide association study (GWAS) can be used to detect mutations associated with Mtb drug resistance. A first GWAS (n=127) attempted to identify mutations associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. A second GWAS was applied to a large global set (n>6400) to identify mutations associated with first- and secondline drug resistance. M. aurum is an environmental mycobacteria that has been proposed as a model for the development of anti-TB drugs. I have assembled and annotated its draft genome, and identified copy number variants in known drug resistance targets. Approximately 10% of the Mtb genome consists of two gene families (pe/ppe) that are poorly characterised, and are hypothesised to be important virulence factors. Using a de novo assembly approach, I characterised these genes and their diversity across a global collection of clinical isolates with high depth short-read sequence data (n=518). A follow-up study using a long-read sequence technology (n=18, diverse stain types) confirmed the findings. This work also generated new annotated reference genomes and characterised methylation sites, which may affect transmissibility, pathogenicity and virulence. A future direction of the TB genomics field is to identify genetic check points in host-pathogen interactions using both human and Mtb genotypes. I analysed the genomes of ~720 TB case–Mtb pairs and identified susceptibility markers, which are promising targets for future control measures.

توانمندی تشخیص و ردیابی صحیح فیتوپلاسماها از گیاهان، اهمیت به سزایی در کنترل موثر این عوامل و جلوگیری از گسترش آنها از طریق مواد گیاهی آلوده دارد. استخراج DNA به گونه ای که کمترین مواد ممانعت کننده و بیشترین میزان ماده ژنتیکی فیتوپلاسما حاصل گردد، در موفقیت آزمون واکنش زنجیره ای پلیمراز و صحت نتایج حاصل از آن تاثیرگذار است. در این تحقیق، مقایسه روش های مختلف استخراج DNA نشان داد که استفاده از روش مناسب، در کاهش واکنش منفی کاذب اهمیت دارد. اما حتی در روش موفق استخراج DNA مبتنی بر استفاده از ستون نیز واکنش های منفی کاذب هرچند به تعداد اندک اما به صورت پراکنده وجود دارد. این امر می تواند ناشی از غلظت پایین و پراکنش نامنظم سلول-های فیتوپلاسمایی در بافت‌های گیاه میزبان باشد. بر اساس نتایج حاصل از تعیین توالی قطعه تکثیر شده با ترکیب آغازگری P1/P7-R16F2n/R16R2، واکنش مثبت دروغین در برخی تکرارهای آزمون Nested PCR به دست آمد. به منظور کاهش آلودگی های متقاطع و اجتناب از واکنش های مثبت دروغین، آزمون Single Tube Nested PCR بهینه سازی گردید. علیرغم دستیابی به محاسن مختلف این روش از جمله سهولت اجرا، صرفه جویی در هزینه و زمان و همچنین توان ردیابی بیمارگر در غلظت های پایین، متاسفانه واکنش های مثبت کاذب در تعداد کمی از نمونه ها همچنان مشاهده شد. در مجموع نتایج حاصل از روش Nested PCR با یک ترکیب آغازگری و به تنهایی برای ردیابی و ارزیابی-های وسیع باغات و مزارع کفایت نمی کند و استفاده از ترکیبات پرایمری مختلف، تعیین توالی و یا هضم آنزیمی به منظور اجتناب از واکنش مثبت دروغین توصیه می گردد.

Chromosome 21 is the smallest human chromosome and represents a model for physical mapping of the entire human genome. Three copies of this autosome cause Down syndrome, the most frequent genetic disorder associated with significant mental retardation. Five years ago, in the framework of the international human genome project, and as an integrated part of the chromosome 21 community, a consortium, of academic groups from Japan and Germany was formed to map and sequence this chromosome. Several whole-genome and chromosome-specific DNA libraries were constructed for mapping purposes. Using a combination of nested-deletion [1] and shotgun sequencing approaches, our center determined and analyzed over 17 million bases of high-quality data. In total, over 33.5 million basepairs of DNA, distributed over four contigs, were sequenced with very high accuracy [2]. The largest of these contigs is nearly 25.5Mb long. Only three small clone gaps remain, which together comprise approximately 100kb. Thus, we achieved a coverage of 99.7% of 21q. In addition, 211,116 bp from the short arm were also sequenced. Analysis of the chromosome revealed far fewer genes than previously estimated. Instead of 800-1000 expected genes, we found only 225 genes, of which 127 are known genes and 98 are predicted genes. In addition, 59 pseudogenes were identified. The completion of the 21q sequence provides a unique resource for understanding the molecular pathophysiology of Down syndrome, as well as all other monogenic and complex disorders that map to this chromosome, including Alzheimer’s disease, leukemia, autoimmune disease, epilepsy and manic-depressive psychosis. It also stands as a structural framework from which the complete molecular architecture of the chromosome can be determined. With the announcement of the first “working draft” of the human genome by the international human genome sequencing consortium this past June, there has been a great interest in the total number of genes in the human genome[3]. Some of the current estimates range from as low as 34,000 genes to as high as 140,000 genes. The low gene-density of chromosome 21 was quite unexpected. Most striking is a 7-Mb region near the region near the centromere that contains only one gene. This region is much larger than the whole genome of some species, such as Escherichia coli, yet evolutionary processes permitted the existence of such a gene-poor SNA segment. This finding leads us to propose that there are additional large gene-less regions in other human/mammalian chromosomes. By combining the gene numbers of chromosomes 21 and 22 [4] (770genes; 2-3% of human genome) and assuming that together they represent the average gene content of the human genome, we estimate that the total number of human genes may be close to 40,000. As part of the international human genome sequencing project, we are also sequencing parts of chromosomes 11 and 18. Our high-throughput sequence production line generates about 10 Mb per day. In order to handle such a high volume of output, we have developed a system for automated data assembly, annotation and release. All the data is released through our website and through the DNA Database of Japan immediately according to the policies established by the international consortium. In addition to chromosomes 11, 18 and 21, we are also interested in the structure of the entire human genome. In collaboration with the University of Tokyo, we have developed a database called HGREP [5] (Human Genome Reconstruction Project) that contains working draft and finished sequences which cover more than 85% of the human genomic sequences. Sequence entries are aligned along the chromosomes based on sequence similarities to STS markers, BAC-end and other entry sequences. Further, biological features, such as genes, gene functions, repeats and CpG islands, are fully annotated on these entries. To identify evolutionally conserved and biologically important information in the genome, we are taking the strategy of comparative genomic sequence analysis between human and other related genomes such as those of rodents and primates. We are currently sequencing several regions of the mouse, including some regions that are counterpart to human chromosome 21, and are preparing genomic resources for sequencing of the chimpanzee. Through comparative methods, we are also trying to understand telomeric regions, structures at the end of chromosomes that play an important role in several biological functions and have been associated with human diseases like an oncogenesis and a cellular aging. The structure and function of all genes and their regulatory regions can only be fully understood by looking at how they interact under different circumstances and in several different backgrounds. Determining which genes and elements are human-specific will lead to a deeper understanding of who we are and will advance medical sciences drastically.

Objective To study the effects of different pre-sequencing sample processing modes on the results of whole genome sequencing with high-throughput sequencing (HTS) by taking the largest RNA virus (human coronavirus, HCoV) as the representative. Methods Cell-cultured human coronavirus HCoV-OC43 strains were used as the representative samples and divided into different groups based on pre-sequencing processing modes as follows: untreated group, DNase and RNase treatment before nucleic acid extraction group, DNase treatment after nucleic acid extraction group, and DNase and RNase treatment before nucleic acid extraction and DNase treatment after nucleic acid extraction group. Nucleic acid samples of each group were analyzed by direct RNA sequencing (without amplification) and DNA sequencing after sequence independent single primer amplification (SISPA), respectively. Results No significant difference in viral genome coverage rates was observed between different groups. The highest genome coverage and sequencing accuracy were obtained in DNase treatment after nucleic acid extraction group by direct RNA sequencing, and the ratio of viral reads and the sequencing depth of each locus were effectively improved by SISPA amplification. Conclusions This study provided an optimized technical strategy for whole genome sequencing of RNA viruses such as coronavirus. Key words: Human coronavirus; Sequence independent single primer amplification; Whole genome sequencing; High-throughput sequencing

Objective To determine whether a periprosthetic joint infections (PJI) patient derived S. aureus is an appropriate candidate tool bacterium for studying the pathogenesis of S. aureus-induced PJI. Methods Whole genome sequencing data of ST1792 was obtained using the Illumina Hiseq platform of a Staphylocuccal. aureus(S. aureus) strain derived from a PJI prosthesis, and gene functional enrichment analysis based on gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) database was performed. Besides, its biofilm formation ability in drainage fluid, synovial fluid and human plasmin were compared to that in experimental cultures, in order to determine differences in biofilm forming ability in different environment. In addition, based on Illumina Hiseq platform, transcriptome analysis of the S. aureus in the three types of fluid above was performed to determine its gene expression profile and functional enrichment analysis of the differentially expressed genes was conducted through the GO and KEGG database. Finally, adaptability in different human fluid of the S. aureus was analyzed with the transcriptome profile and biofilm formation data. Results The genome of ST1792 consisted of 2 718 coding sequences, including main virulence encoding genes and regulation related genes. Functional enrichment showed no sign of significant missing or major recombination in the bacterial genome. The biofilm mass in plasmin was much higher than that in synovial fluid, both significantly higher than the biofilm mass in TSB. But the biofilm mass in drainage fluid was not statistically different from that in TSB. This revealed that the bacterial biofilm phenotype was significantly altered in PJI human fluid. Transcriptomics sequencing revealed that a huge number of genes were differentially expressed in drainage fluid, synovial fluid and human plasmin compared to TSB. Functional enrichment revealed that the bacterial gene expression profile was different in PJI human fluids, which showed the sophisticated adaptation of the bacterium to human fluid environment. Conclusion As a clinical S. aureus isolated from a PJI patient, ST1792 presents distinct phenotypes and adaptations in vivo, so it is a proper candidate for future studies in the pathogenesis of S. aureus induced PJI. Key words: Biofilms; Prosthesis-related infections; Staphylococcus aureus; Genomics

Objective To investigate the pathogenic mechanism of Campylobacter jejuni(C.jejuni) associated with Guillain-Barre syndrome(GBS) and provide strategy for gene modification, the cstⅡ gene from 8 GBS-associated C.jejuni strains were compared with that from 3 GBS-unrelated C.jejuni strains, getting the base and amino acid mutations, the changes of secondary structures and finding the region which may be responsible for the pathogenicity of C.jejuni inducing GBS. Methods Three GBS-associated C.jejuni strains isolated from stools of GBS patients in north China were selected and cultured, which has been confirmed as GBS-associated by animal model. After sequencing the genome of them, the nucleotide sequences of cstⅡ gene were got through sequence alignment. The nucleotide sequences and deduced amini acid sequences of 3 GBS-associated cstⅡ genes were compared with that from 3 GBS-unrelated C.jejuni strains through bioinformatics software, getting the base and amino acid mutations, the changes of secondary structures. Other 5 GBS-associated cstⅡ genes were also aligned to know whether the differences we got above makes sense. In this way the genetic differences between two kinds of C.jejuni strains may be found and speculating the gene region related to the pathogenicity of GBS became possible. Results The cstⅡgene of 3 GBS-associated C.jejuni strains were all composed of 876 base pairs. Compared with GBS-unrelated C.jejuni strains, there were 9 consistent mutation sites in cstⅡ gene of LL and QYT stains, leading to 3 consistent amino acid mutation. The amino acid mutation of 114 and 182 sites in LL and QYT stains existed in other 5 GBS-associated C.jejuni strains. The sole amino acid mutation of ZHX strain -169 site, located near the 182 site. The seventh α-helix(165-180 region)of the secondary structure of the amino acid sequence from GBS-associated strains were shorter than that from GBS-unrelated strains, and the shorter regions were opened to form β-sheet or coli, which also existed in other GBS-related strains in this study.75% of the GBS-associated cstⅡ genes were Asn-51, while 25% of the GBS-associated and all of the GBS-unrelated cstⅡ genes were Thr-51.LL strain showed highly identity to other GBS-unrelated strains in this study. Conclusion The 165-180 segment of secondary structures in cstⅡ gene from local 3 GBS-associated C.jejuni strains are probably the responsible region involved in inducing GBS. The senior structure changes in this region may affect the activity of sialyltransferase and the structures of ganglioside epitope, so that the C.jejuni can acquire the pathogenicity of GBS. This finding may give a clue to genetic modified site. The bi-functional cstⅡ of C.jejuni may be related to the pathogenicity of GBS. The cstⅡ of LL strain to some extent represents the characteristics of Asian strains, which may directs strains monitoring. Key words: Guillain-Barre syndrome; Campylobacter jejuni; cstⅡ gene; Sequence analysis; Secondary structure

Recent advances in High-Throughput Sequencing (HTS) technology have greatly facilitated the researches in bioinformatics field. With the ultra-high sequencing speed and improved base-calling accuracy, Illumina Genome Analyzer is currently the most widely used platform in the field. To use the raw reads generated from the sequencing machine, the 3’ adapter sequence attached to the real read in the process of ligation needs to be correctly trimmed. This is often done by some inhouse scripts or different packages with various parameters. They either use the Smith-Waterman algorithm or search for an exact match of the 3’ adapter sequence. In this report, I investigated methodologies as well as the strengths and weaknesses of five representative mainstream adapter trimming tools in order to suggest a direction for other researchers. Furthermore, four sets of detailed analysis were performed to evaluate the performances of these tools. I demonstrated that my adapter trimming method is flexible, accurate and efficient for Next Generation Sequencing (NGS) analysis. Type of Report: MS Project Report Department of Computer Science & Engineering Washington University in St. Louis Campus Box 1045 St. Louis, MO 63130 ph: (314) 935-6160 Master’s Project Report LCScanner: An Efficient and Accurate Trimming Tool for Illumina Next Generation Sequencing Reads by Xiang Zhou Master of Engineering in Computer Science Washington University in St. Louis, 2012 Research Advisor: Professor Weixiong Zhang

Objective To investigate the sequence variations of mitochondrial D-loop region in chronic glomerular nephritis patients and family members and their possible associations with chronic glomerular nephritis. Methods 20 patients with chronic glomerular nephritis and 48 unaffected pedigree members, as well as 122 cases of normal control were recruited.mtDNA extracted from peripheral blood were examined by PCR-based assay for D-loop sequence variations, followed by sequencing analysis. Results Compared with the normal control and standard sequence, a total of 61 sequence variants were detected, among these, three high variations were found, and the base variation rate of patients with chronic glomerular nephritis (CGN) and unaffected pedigree members(UAPM)(0.88%, 0.72%) significantly increased compared with the control group(0.61%). The distibutional difference of base variation rate in the experimental groups were significantly higher than those in the control group(χ2=21.220, 7.964, all P<0.05). Especially in microsatellite variation in D310 region, the mutation frequency and base variation rate in patients with CGN were 100.00% and 30.00%, respectively, which of UAPM were 81.30% and 17.95%, respectively, while those in the normal control group were 53.30% and 6.05%, respectively.Among them, the patients with CGN compared with the control group had statistically significant differences (χ2=15.610, 150.047, all P<0.05). Likewise, UAPM also had statistically significant difference compared with the control group(χ2=11.347, 66.188, all P<0.05). Conclusion D-Loop of mitochondrial genome mutations may be related to the development of chronic glomerular nephritis. Key words: DNA, mitochondrial; Glomerulonephritis; Mutation; Microsatellite instability

Whole-genome exon sequencing technology is a genomic analysis method that uses target sequence capture technology to capture DNA from all exon regions of the genome for high-throughput sequencing, and has a high sensitivity for identifying disease-related low-frequency and rare mutations. Type 2 diabetes mellitus is characterized by progressive islet β-cell dysfunction and insulin resistance, and it is thought to be the result of a combination of genetic and environmental factors, in which genetic factors play an important role in the pathogenesis of type 2 diabetes. This article reviews the application of whole-genome exon sequencing in the screening of susceptibility genes and coding variants in type 2 diabetes mellitus. Key words: Exome sequencing; Diabetes mellitus, Type2; Gene mutation

The primary structure and the coding capacity of the insect iridescent virus type 6--Chilo iridescent virus (CIV)--were determined between the genome coordinates 0.310 (EcoRI site) and 0.347 (ClaI site). The EcoRI CIV DNA fragment M (7.1 kb; 0.310-0.345 map units) harbors one out of at least six loci of DNA replication origins which is located at nucleotide position 485-513. The identification of the structural properties and the coding capacity of the EcoRI CIV DNA fragment M was carried out by DNA nucleotide sequencing, computer-aided sequence analysis and DNA/RNA hybridization. The EcoRI CIV DNA fragment M (7,099 bp; 71.14% A+T and 28.86% G+C) possesses two clusters of five tandemly organized repetitive DNA elements with complex structural arrangements (R1-R5) which are located between nucleotide positions 3272-3350 and 3403-3414. The analysis of the DNA sequences of the EcoRI CIV DNA fragment M revealed the presence of six open reading frames (ORFs 1-6). Two out of six detected putative proteins are of particular interest. ORF-2 was found to be terminated at nucleotide position 366 (TAA) within the DNA sequence of the EcoRI CIV DNA fragment L (0.345-0.381 map units; 7.4 kb). The analysis of ORF-2 (1,051 amino acids; 120 kD) revealed homologies to several DNA-directed RNA polymerases. ORF-6 encodes a protein (606 amino acids; 69 kD) which is related to a group of yeast, Drosophila and mammalian proteins of a distinct family of putative DNA and/or RNA helicases belonging to the 'DEAD/H' superfamily. The transcriptional activity of the EcoRI CIV DNA fragment M was determined by DNA/RNA hybridization experiments. These analyses revealed the existence of three RNA transcripts of about 3.4 kb (t1), 1.8 kb (t2) and 1.2 kb (t3) which agree with the predicted size of the expected RNA transcripts from ORF-2 (1,051 amino acids; 3.1 kb) and ORF-6 (606 amino acids; 1.8 kb).

Characterization of the cytochrome [beta] gene in plant pathogenic, basidiomycetes and consequences for QoI resistance

Objective To study the structural features and different types of the genomic molecular organization of human bocavirus in infected infants in Guangzhou. Methods Primers were designed on the basis of the genomic sequence of human bocavirus BJ3064 strain (DQ988933) in the GenBank, and amplify HBOV genome by PCR.The PCR-products were directly sequenced. The genomic nucleotide sequences were analyzed with the programs of CLUSTAL W/X and MEGA5.0.Results The strain GZ2010-1 genome is 5299bp in length. The genomic sequences of GZ2010-1 strain were compared with those sequences of HBOV1, HBOV2, HBOV3, and HBOV4 genotypes in GenBank, revealed that the homology with HBOV1 genotype ranged from 98% to 99%, homology with genotypes HBOV2, HBOV3, HBOV4 were 76%, 81% ,and 75% respectively. The F genes of GZ2010-1 strain was compared with F genes of HBOV1 genotype, showed that the homology was also between 98%-99%. Conclusions The complete nucleotide sequence of human bocavirus GZ2010-1 strain isolated from Guangzhou is 5299 bp in length,GenBank accession NO.is JN 128956. Sequence and phylogenetic analysis of the GZ2010-1 strain reveales that this isolate belongs to HBOV1. Key words: Human bocavirus; Genome; Sequence analysis; Guangzhou

Non-synonymous cSNPs that occurred in the protein-coding region could potentially alter the amino acid composition and thus the functionality of gene product. Protein variants generated by these non-synonymous cSNPs are thought to be responsible for the diversity of human population and the cause of human diseases associated with inheritance. It is essential to identify these non-synonymous cSNPs/functional variants for functional genomic studies. In addition to direct genome sequencing methods, dbEST has been used for SNP discovery due to its rich information of millions ESTs. Previous SNP discovery methods from dbEST needed to include many ESTs per cluster, which greatly reduce the effectiveness of dbEST. Such approaches would be useful in identifying SNPs for polymorphic markers. We would like to identify these sequence variations as either disease association mutations or functional variants. Therefore, it is necessary to use alternative bioinformatic tools to extract these low frequency functional variants. We have used our CGI bioinformatic tools to data-mining functional variants from ESTs, especially low frequency non-synonymous cSNPs. We have established the CGI tools for comparative genomic studies in identifying novel human genes. With 9848 human reference protein as starting alignment scaffolds and 2.2 million human ESTs, we have now identified more than 50,000 potential functional variants. In addition, our approach can be used to validate and correct amino acid sequences of human reference proteins.