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Targeted transcript profiling by sequencing
KTH, School of Biotechnology (BIO), Gene Technology. (Gene technology)
KTH, School of Biotechnology (BIO), Gene Technology. (Gene technology)ORCID iD: 0000-0003-4313-1601
KTH, School of Biotechnology (BIO), Gene Technology. (Gene technology)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In recent years, second generation sequencers have been employed to study various facets of the transcriptome in a comprehensive manner. However, intermediary gene sets featuring differentially expressed genes can reduce the dimensionality of experiments while providing researchers with the most significant data. Trinucleotide threading (TnT) is a multiplex amplification method previously implemented in an assay for expression profiling of moderate gene sets. Here, two additional detection systems were evaluated with a focus on lowering the input material requirements. 32 genes were simultaneously assayed with detection either by direct hybridization of TnT products or by sequencing these using the massively parallel 454 sequencer. Both approaches produced reliable transcript abundance data starting from total RNA from about 200 cells. The direct hybridization readout is beneficial for smaller-scale studies, while more ambitious efforts employing numerous individuals are, together with a sample barcoding and pooling scheme, well suited for the second generation sequencing approach. Moreover, with protocol optimizations the starting material requirements for the sequencing strategy may be further reduced. Accordingly, this study presents a targeted RNA-Seq method.

National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11293OAI: oai:DiVA.org:kth-11293DiVA: diva2:272179
Note
QC 20100819Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2011-02-04Bibliographically approved
In thesis
1. 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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