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Expression profiling of signature gene sets with trinucleotide threading
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0003-4313-1601
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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.

Place, publisher, year, edition, pages
2008. Vol. 9, no 2, 209-217 p.
Keyword [en]
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: urn:nbn:se:kth:diva-7689DOI: 10.1016/j.ygeno.2007.10.012ISI: 000252944500011Scopus ID: 2-s2.0-38149086782OAI: oai:DiVA.org:kth-7689DiVA: diva2:12789
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-08-13Bibliographically approved
In thesis
1. Interrogation of Nucleic Acids by Parallel Threading
Open this publication in new window or tab >>Interrogation of Nucleic Acids by Parallel Threading
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
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:nbn:se:kth:diva-4546 (URN)978-91-7178-802-3 (ISBN)
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
2. Parallel target selection by trinucleotide threading
Open this publication in new window or tab >>Parallel target selection by trinucleotide threading
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 91 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:19
Keyword
trinucleotide threading, multiplex amplification, expression profiling, microarray, generic tag, short tandem repeat, microsatellite, electrophoresis, single nucleotide polymorphism, allelotyping, 454, Pyrosequencing
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11284 (URN)978-91-7415-431-3 (ISBN)
Public defence
2009-11-06, FR4 (Oscar Kleins Auditorium), AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:30 (Swedish)
Opponent
Supervisors
Note
QC 20100819Available from: 2009-10-15 Created: 2009-10-13 Last updated: 2011-11-23Bibliographically approved

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