Improving Short DNA Sequence Alignment with Parallel Computing

Variations in different types of genomes have been found to be responsible for a large degree of physical diversity such as appearance and susceptibility to disease. Identification of genomic variations is difficult and can be facilitated through computational analysis of DNA sequences. Newly available technologies are able to sequence billions of DNA base pairs relatively quickly. These sequences can be used to identify variations within their specific genome but must be mapped to a reference sequence first. In order to align these sequences to a reference sequence, we require mapping algorithms that make use of approximate string matching and string indexing methods. To date, few mapping algorithms have been tailored to handle the massive amounts of output generated by newly available sequencing technologies. In otrder to handle this large amount of data, we modified the popular mapping software BWA to run in parallel using OpenMPI. Parallel BWA matches the efficiency of multithreaded BWA functions while providing efficient parallelism for BWA functions that do not currently support multithreading. Parallel BWA shows significant wall time speedup in comparison to multithreaded BWA on high-performance computing clusters, and will thus facilitate the analysis of genome sequencing data.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

Complete Genome Sequence of Erwinia amylovora Bacteriophage

The complete genome of an Erwinia amylovora bacteriophage, vB_EamM_Ea35-70 (Ea35-70), is 271,084 bp, encodes 318 putative proteins, and contains one tRNA. Comparative analysis with other Myoviridae genomes suggests that Ea35-70 is related to the Phikzlikevirus genus within the family Myoviridae, since 26% of Ea35-70 proteins share homology to proteins in Pseudomonas phage φKZ.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

Molecular cloning restriction enzyme analysis, bovine adenovirus type 2 genome

Adenoviruses are non-enveloped icosahedral-shaped particles which possess a double-stranded DNA genome. Currently, nearly 100 serotypes of adenoviruses have been identified, 48 of which are of human origin. Bovine adenoviruses (BAVs), causing both mild respiratory and/or enteral diseases in cattle, have been reported in many countries all over the world. Currently, nine serotypes of SAVs have been isolated which have been placed into two subgroups based on a number of characteristics which include complement fixation tests as well as the ability to replicate in various cell lines. Bovine adenovirus type 2 (BAV2), belonging to subgroup I, is able to cause pneumonia as well as pneumonic-like symptoms in calves. In this study, the genome of BAV2 (strain No. 19) was subcloned into the plasmid vector pUC19. In total, 16 plasmids were constructed; three carry internal San fragments (spanning 3.1 to 65.2% ), and 10 carry internal Pstl fragments (spanning 4.9 to 97.4%), of the viral genome. Each of these plasmids was analyzed using twelve restriction endonucleases; BamHI, CiaI, EcoRl, HiOOlll, Kpnl, Noll, NS(N, Ps~, Pvul, Saj, Xbal, and Xhol. Terminal end fragments were also cloned and analyzed, sUbsequent to the removal of the 5' terminal protein, in the form of 2 BamHI B fragments, cloned in opposite orientations (spanning 0 to 18.1°k), and one Pstll fragment (spanning 97.4 to 1000/0). These cloned fragments, along with two other plasmids previously constructed carrying internal EcoRI fragments (spanning 20.6 to 90.5%), were then used to construct a detailed physical restriction map using the twelve restriction endonucleases, as well as to estimate the size of the genome for BAV2(32.5 Kbp). The DNA sequences of the early region 1 (E1) and hexon-associated gene (protein IX) have also been determined. The amino acid sequences of four open reading frames (ORFs) have been compared to those of the E1 proteins and protein IX from other Ads.

The identification and characterization of inter- and intra-species genetic diversity derived from retrotransposons in humans

Retrotransposons, which used to be considered as “junk DNA”, have begun to reveal their immense value to genome evolution and human biology due to recent studies. They consist of at least ~45% of the human genome and are more or less the same in other mammalian genomes. Retrotransposon elements (REs) are known to affect the human genome through many different mechanisms, such as generating insertion mutations, genomic instability, and alteration in gene expression. Previous studies have suggested several RE subfamilies, such as Alu, L1, SVA and LTR, are currently active in the human genome, and they are an important source of genetic diversity between human and other primates, as well as among humans. Although several groups had used Retrotransposon Insertion Polymorphisms (RIPs) as markers in studying primate evolutionary history, no study specifically focused on identifying Human-Specific Retrotransposon Element (HS-RE) and their roles in human genome evolution. In this study, by computationally comparing the human genome to 4 primate genomes, we identified a total of 18,860 HS-REs, among which are 11,664 Alus, 4,887 L1s, 1,526 SVAs and 783 LTRs (222 full length entries), representing the largest and most comprehensive list of HS-REs generated to date. Together, these HS-REs contributed a total of 14.2Mb sequence increase from the inserted REs and Target Site Duplications (TSDs), 71.6Kb increase from transductions, and 268.2 Kb sequence deletion of from insertion-mediated deletion, leading to a net increase of ~14 Mb sequences to the human genome. Furthermore, we observed for the first time that Y chromosome might be a hot target for new retrotransposon insertions in general and particularly for LTRs. The data also allowed for the first time the survey of frequency of TE insertions inside other TEs in comparison with TE insertion into none-TE regions. In summary, our data suggest that retrotransposon elements have played a significant role in the evolution of Homo sapiens.

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.