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Interrogation of Nucleic Acids by Parallel Threading
KTH, School of Biotechnology (BIO).
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Advancements in the field of biotechnology are expanding the scientific horizon and a promising era is envisioned with personalized medicine for improved health. The amount of genetic data is growing at an ever-escalating pace due to the availability of novel technologies that allow massively parallel sequencing and whole-genome genotyping, that are supported by the advancements in computer science and information technologies. As the amount of information stored in databases throughout the world is growing and our knowledge deepens, genetic signatures with significant importance are discovered. The surface of such a set in the data mining process may include causative- or marker single nucleotide polymorphisms (SNPs), revealing predisposition to disease, or gene expression signatures, profiling a pathological state. When targeting a reduced set of signatures in a large number of samples for diagnostic- or fine-mapping purposes, efficient interrogation and scoring require appropriate preparations. These needs are met by miniaturized and parallelized platforms that allow a low sample and template consumption.

This doctoral thesis describes an attempt to tackle some of these challenges by the design and implementation of a novel assay denoted Trinucleotide Threading (TnT). The method permits multiplex amplification of a medium size set of specific loci and was adapted to genotyping, gene expression profiling and digital allelotyping. Utilizing a reduced number of nucleotides permits specific amplification of targeted loci while preventing the generation of spurious amplification products. This method was applied to genotype 96 individuals for 75 SNPs. In addition, the accuracy of genotyping from minute amounts of genomic DNA was confirmed. This procedure was performed using a robotic workstation running custom-made scripts and a software tool was implemented to facilitate the assay design. Furthermore, a statistical model was derived from the molecular principles of the genotyping assay and an Expectation-Maximization algorithm was chosen to automatically call the generated genotypes. The TnT approach was also adapted to profiling signature gene sets for the Swedish Human Protein Atlas Program. Here 18 protein epitope signature tags (PrESTs) were targeted in eight different cell lines employed in the program and the results demonstrated high concordance rates with real-time PCR approaches. Finally, an assay for digital estimation of allele frequencies in large cohorts was set up by combining the TnT approach with a second-generation sequencing system. Allelotyping was performed by targeting 147 polymorphic loci in a genomic pool of 462 individuals. Subsequent interrogation was carried out on a state-of-the-art massively parallelized Pyrosequencing instrument. The experiment generated more than 200,000 reads and with bioinformatic support, clonally amplified fragments and the corresponding sequence reads were converted to a precise set of allele frequencies.

Place, publisher, year, edition, pages
Stockholm: KTH , 2007. , 46 p.
Keyword [en]
genotyping, multiplex amplification, trinucleotide threading, single nucleotide polymorphism, genotype calling, Expectation-Maximization, protein-epitope signature tag, expression profiling, Human Protein Atlas, pooled genomic DNA, Pyrosequencing, 454, allelotyping, association studies, bioinformatics
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4546ISBN: 978-91-7178-802-3 (print)OAI: oai:DiVA.org:kth-4546DiVA: diva2:12791
Public defence
2007-12-14, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-08-13Bibliographically approved
List of papers
1. Tri-nucleotide Threading for parallel amplification of minute amounts of genomic DNA
Open this publication in new window or tab >>Tri-nucleotide Threading for parallel amplification of minute amounts of genomic DNA
2006 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 6, 9- p.Article in journal (Refereed) Published
Abstract [en]

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

Keyword
genomic DNA; trinucleotide; cytidine triphosphate; guanosine triphosphate; pyrimidine nucleotide; thymidine 5'-triphosphate; thymidine triphosphate; article; cancer; computer program; DNA microarray; gene amplification; gene locus; genetic variability; genotype; high throughput screening; human; phenotype; priority journal; scoring system; sensitivity and specificity; single nucleotide polymorphism; chemistry; evaluation; genome; genomics; methodology; polymerase chain reaction; Cytidine Triphosphate; Genome, Human; Genomics; Genotype; Guanosine Triphosphate; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Software; Thymine Nucleotides
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7687 (URN)10.1093/nar/gkl103 (DOI)000237002900005 ()2-s2.0-33645832164 (Scopus ID)
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
2. Classification of SNP genotypes by a Gaussian mixture model
Open this publication in new window or tab >>Classification of SNP genotypes by a Gaussian mixture model
Show others...
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7688 (URN)
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-08-13Bibliographically approved
3. Expression profiling of signature gene sets with trinucleotide threading
Open this publication in new window or tab >>Expression profiling of signature gene sets with trinucleotide threading
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2008 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 9, no 2, 209-217 p.Article in journal (Refereed) Published
Abstract [en]

In recent years, studies have shown that expression profiling of carefully chosen intermediary gene sets, comprising approximately 10 to 100 genes, can convey the most relevant information compared to much more complex whole-genome studies. In this paper, we present a novel method suitable for expression profiling of moderate gene sets in a large number of samples. The assay implements the parallel amplification features of the trinucleotide threading technique (TnT), which encompasses linear transcript-based DNA thread formation in conjunction with exponential multiplexed thread amplification. The amplifications bestow the method with high sensitivity. The TnT procedure together with thread detection, relying on thread-specific primer extension followed by hybridization to universal tag arrays, allows for three distinction levels, thus offering high specificity. Additionally, the assay is easily automated and flexible. A gene set, comprising 18 protein epitope signature tags from the Swedish Human Protein Atlas program, was analyzed with the TnT-based approach and the data were compared with those generated by both real-time PCR and genome-wide cDNA arrays, with the highest correlation observed between TnT and real-time PCR. Taken together, expression profiling with trinucleotide threading represents a reliable approach for studies of intermediary gene sets.

Keyword
Gene expression profiling; HPA; Microarray; Multiplex; Real-time PCR; TnT; complementary DNA; DNA polymerase; epitope; transcriptome; trinucleotide; article; cell line; controlled study; DNA synthesis; gene expression profiling; housekeeping gene; human; human cell; microarray analysis; priority journal; real time polymerase chain reaction; RNA isolation; serial analysis of gene expression; signal noise ratio; DNA Primers; Epitopes; Gene Expression Profiling; Humans; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7689 (URN)10.1016/j.ygeno.2007.10.012 (DOI)000252944500011 ()2-s2.0-38149086782 (Scopus ID)
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
4. Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
Open this publication in new window or tab >>Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
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2008 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, 323-329 p.Article in journal (Refereed) Published
Abstract [en]

Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

Keyword
trinucleotide threading; pooled genomic DNA; association studies; multiplexing; SNP; allelotyping; cancer; obesity
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7690 (URN)10.1002/humu.20655 (DOI)000253033000016 ()2-s2.0-38949145121 (Scopus ID)
Note
QC 20100416Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved

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