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Phasing of single DNA molecules by massively parallel barcoding
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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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.

Place, publisher, year, edition, pages
2015. Vol. 6, 7173
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-171312DOI: 10.1038/ncomms8173ISI: 000357166400001PubMedID: 26055759Scopus ID: 2-s2.0-84931275307OAI: oai:DiVA.org:kth-171312DiVA: diva2:843142
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
In thesis
1. Technologies for Single Cell Genome Analysis
Open this publication in new window or tab >>Technologies for Single Cell Genome Analysis
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
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:nbn:se:kth:diva-181059 (URN)978-91-7595-842-2 (ISBN)
Public defence
2016-02-19, Air and Fire, Science for Life Laboratory, KTH, Tomtebodavägen 23A, Solna, 10:00 (English)
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Supervisors
Note

QC 20160127

Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2016-01-27Bibliographically approved

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