23 research outputs found
Flow cytometry for enrichment and titration in massively parallel DNA sequencing
Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols
Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates
Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples
Methods for Analyzing Genomes
The human genome reference sequence has given us a twoâdimensional blueprint of our inherited code of life, but we need to employ modernâday technology to expand our knowledge into a third dimension. Interâindividual and intraâindividual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNAâsequencing platforms has increased a thousandâfold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions. Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and costâefficient method for enrichment and titration of clonally amplified DNAâlibraries on beads. The method uses fluorescent labeling and a flowâcytometer to separate DNAâbeads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sunâexposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its wellâdocumented involvement in the disease progression of cancers, an estimated 35,000 novel sunâinduced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor proteinâaltering mutations in multiple individuals
Methods for Analyzing Genomes
The human genome reference sequence has given us a twoâdimensional blueprint of our inherited code of life, but we need to employ modernâday technology to expand our knowledge into a third dimension. Interâindividual and intraâindividual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNAâsequencing platforms has increased a thousandâfold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions. Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and costâefficient method for enrichment and titration of clonally amplified DNAâlibraries on beads. The method uses fluorescent labeling and a flowâcytometer to separate DNAâbeads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sunâexposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its wellâdocumented involvement in the disease progression of cancers, an estimated 35,000 novel sunâinduced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor proteinâaltering mutations in multiple individuals
A Novel Method for Rapid Hybridization of DNA to a Solid Support
<div><p>Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.</p></div
Principle of MFH in genotyping combined with Multiplex Competitive Hybridization (MUCH).
<p>MUCH-probes are designed to target all genotypes of interest. Each probe consists of two parts, one target specific region (1,3) that will hybridize and recognize a particular genotype and a corresponding address tag, designed to be orthogonal i.e. as different as possible as compared to other address tags in the pool. When (1,3) may differ at 3â5 bp positions, (2,4) are as different as possible to each other. The PCR-product (6) has been coupled to the bead (5) and made single stranded. The bead-product solution is mixed with tagged MUCH probes which are allowed to hybridize to the single-stranded amplicon following rapid heating of the sample (a). After successful MUCH, the bead-product-MUCH-tag containing solution is deposited (b) onto the detection array surface (7) and is subjected to magnetic forced hybridization using a neodymium magnet under the detection surface (10). The bead-product-MUCH-tag will hybridize via MFH to its complementary oligo-tag on the array (8). The results are readily visible through visual inspection (c) and the sample is genotyped as variant (3).</p
HPV diagnostic assay.
<p>a) Theoretical detection pattern for the HPV diagnostic assay for HPV 18 and HPV 45, and actual results after MFH documented with b) an array scanner and c) a digital camera. The MUCH assay requires both interrogation probes for a particular HPV species to give a positive signal, in order to give positive call for the HPV species.</p
Principle of Magnetic Forced Hybridization (MFH).
<p>A double stranded PCR-product (1) carries a PCR-primer incorporated biotin affinity molecule (2). The PCR-product is bound (a) via biotin-streptavidin coupling to magnetic beads (3) and manipulated via alkali treatment (b) to become single stranded. An oligonucleotide array (4) is prepared with reverse complementary sequences (5,6) to targets of interest. The bead-product containing solution is deposited onto the detection array surface (c) and is subjected to magnetic forced hybridization using a neodymium magnet (7) under the detection surface (d). The bead-product hybridizes to its complementary oligo-tag on the array and the results are readily visible through visual inspection (e).</p
Spatial transcriptomics of human placentas reveal distinct RNA patterns associated with morphology and preeclampsia
Spatial transcriptomics (ST) maps RNA level patterns within a tissue. This technology has not been previously applied to human placental tissue. We demonstrate analysis of human placental samples with ST. Unsupervised clustering revealed that distinct RNA patterns were found corresponding to different morphological structures. Additionally, when focusing upon terminal villi and hemoglobin associated structures, RNA levels differed between placentas from full term healthy pregnancies and those complicated by preeclampsia. The results from this study can provide a benchmark for future ST studies in placenta.Susanne Lager and Patrik L. StÄhl are shared senior authors.</p
An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.
Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.QC 20161124</p