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Technologies for Single Cell Genome Analysis
KTH, School of Biotechnology (BIO), Gene Technology.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules.

As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample.

Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. , 48 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:1
Keyword [en]
DNA, sequencing, single molecule, single cell, whole genome amplification, exome sequencing, emulsions, barcoding, phasin
National Category
Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-181059ISBN: 978-91-7595-842-2 (print)OAI: oai:DiVA.org:kth-181059DiVA: diva2:898188
Public defence
2016-02-19, Air and Fire, Science for Life Laboratory, KTH, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20160127

Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2016-01-27Bibliographically approved
List of papers
1. Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations
Open this publication in new window or tab >>Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, e19119- p.Article in journal (Refereed) Published
Abstract [en]

Background: Large efforts have recently been made to automatethe sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. Methodology/Principal Findings: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions. Conclusions/Significance: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.

Keyword
Cell lines
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-33950 (URN)10.1371/journal.pone.0019119 (DOI)000290019400031 ()2-s2.0-79955691833 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20110609Available from: 2011-06-09 Created: 2011-05-23 Last updated: 2017-12-11Bibliographically approved
2. Comparison of Whole Genome Amplification Techniques for Human Single Cell (Exome) Sequencing
Open this publication in new window or tab >>Comparison of Whole Genome Amplification Techniques for Human Single Cell (Exome) Sequencing
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-181056 (URN)
Note

QS 2016

Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2016-01-27Bibliographically approved
3. Transplanted Bone Marrow-Derived Cells Contribute to Human Adipogenesis
Open this publication in new window or tab >>Transplanted Bone Marrow-Derived Cells Contribute to Human Adipogenesis
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2015 (English)In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 22, no 3, 408-417 p.Article in journal (Refereed) Published
Abstract [en]

Because human white adipocytes display a high turnover throughout adulthood, a continuous supply of precursor cells is required to maintain adipogenesis. Bone marrow (BM)-derived progenitor cells may contribute to mammalian adipogenesis; however, results in animal models are conflicting. Here we demonstrate in 65 subjects who underwent allogeneic BM or peripheral blood stem cell (PBSC) transplantation that, over the entire lifespan, BM/PBSC-derived progenitor cells contribute similar to 10% to the subcutaneous adipocyte population. While this is independent of gender, age, and different transplantation-related parameters, body fat mass exerts a strong influence, with up to 2.5-fold increased donor cell contribution in obese individuals. Exome and whole-genome sequencing of single adipocytes suggests that BM/PBSC-derived progenitors contribute to adipose tissue via both differentiation and cell fusion. Thus, at least in the setting of transplantation, BM serves as a reservoir for adipocyte progenitors, particularly in obese subjects.

National Category
Cell Biology Endocrinology and Diabetes
Identifiers
urn:nbn:se:kth:diva-173762 (URN)10.1016/j.cmet.2015.06.011 (DOI)000360453900013 ()26190649 (PubMedID)2-s2.0-84940718419 (Scopus ID)
Funder
Swedish Diabetes AssociationSwedish Cancer SocietySwedish Research CouncilNovo Nordisk
Available from: 2015-09-22 Created: 2015-09-18 Last updated: 2017-12-01Bibliographically approved
4. Phasing of single DNA molecules by massively parallel barcoding
Open this publication in new window or tab >>Phasing of single DNA molecules by massively parallel barcoding
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, 7173Article in journal (Refereed) Published
Abstract [en]

High-throughput sequencing platforms mainly produce short-read data, resulting in a loss of phasing information for many of the genetic variants analysed. For certain applications, it is vital to know which variant alleles are connected to each individual DNA molecule. Here we demonstrate a method for massively parallel barcoding and phasing of single DNA molecules. First, a primer library with millions of uniquely barcoded beads is generated. When compartmentalized with single DNA molecules, the beads can be used to amplify and tag any target sequences of interest, enabling coupling of the biological information from multiple loci. We apply the assay to bacterial 16S sequencing and up to 94% of the hypothesized phasing events are shown to originate from single molecules. The method enables use of widely available short-read-sequencing platforms to study long single molecules within a complex sample, without losing phase information.

National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-171312 (URN)10.1038/ncomms8173 (DOI)000357166400001 ()26055759 (PubMedID)2-s2.0-84931275307 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20150727

Available from: 2015-07-27 Created: 2015-07-27 Last updated: 2017-12-04Bibliographically approved

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