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  • 1.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jacobsson, Josefin
    Fredriksson, Robert
    Marcus, Claude
    Schiöth, Helgi
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads2008In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, p. 323-329Article in journal (Refereed)
    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.

  • 2. Picelli, Simone
    et al.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zhou, Xiao-Lei
    Edler, David
    Lenander, Claes
    Dalen, Johan
    Hjern, Fredrik
    Lundqvist, Nils
    Lindforss, Ulrik
    Pahlman, Lars
    Smedh, Kennet
    Tornqvist, Anders
    Holm, Jorn
    Janson, Martin
    Andersson, Magnus
    Ekelund, Susanne
    Olsson, Louise
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindblom, Annika
    Common variants in human CRC genes as low-risk alleles2010In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 46, no 6, p. 1041-1048Article in journal (Refereed)
    Abstract [en]

    The genetic susceptibility to colorectal cancer (CRC) has been estimated to be around 35% and yet high-penetrance germline mutations found so far explain less than 5% of all cases. Much of the remaining variations could be due to the co-inheritance of multiple low penetrant variants. The identification of all the susceptibility alleles could have public health relevance in the near future. To test the hypothesis that what are considered polymorphisms in human CRC genes could constitute low-risk alleles, we selected eight common SNPs for a pilot association study in 1785 cases and 1722 controls. One SNP, rs3219489:G>C (MUTYH Q324H) seemed to confer an increased risk of rectal cancer in homozygous status (OR = 1.52; CI = 1.06-2.17). When the analysis was restricted to our 'super-controls', healthy individuals with no family history for cancer, also rs1799977:A>G (MLH1 I219V) was associated with an increased risk in both colon and rectum patients with an odds ratio of 1.28 (CI = 1.02-1.60) and 1.34 (CI = 1.05-1.72), respectively (under the dominant model); while 2 SNPs, rs1800932:A>G (MSH6 P92P) and rs459552:T>A (APC D1822V) seemed to confer a protective effect. The latter, in particular showed an odds ratio of 0.76 (CI = 0.60-0.97) among colon patients and 0.73 (CI = 0.56-0.95) among rectal patients. In conclusion, our study suggests that common variants in human CRC genes could constitute low-risk alleles. (C) 2010 Elsevier Ltd. All rights reserved.

  • 3.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Parallel target selection by trinucleotide threading2009Doctoral 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.

  • 4.
    Zajac, Pawel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Targeted transcript profiling by sequencingManuscript (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.

  • 5.
    Zajac, Pawel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gry, Marcus
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Expression profiling of signature gene sets with trinucleotide threading2008In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 9, no 2, p. 209-217Article in journal (Refereed)
    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.

  • 6.
    Zajac, Pawel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Öberg, Christine
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Analysis of Short Tandem Repeats by Parallel DNA Threading2009In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 11, p. e7823-Article in journal (Refereed)
    Abstract [en]

    The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs.

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