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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, article id 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, article id 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, id: 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: 2018-10-02Bibliographically 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. p. 48
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:1
Keywords
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)
Opponent
Supervisors
Note

QC 20160127

Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2016-01-27Bibliographically approved
2. Phasing single DNA molecules with barcode linked sequencing
Open this publication in new window or tab >>Phasing single DNA molecules with barcode linked sequencing
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Elucidation of our genetic constituents has in the past decade predominately taken the form of short-read DNA sequencing. Revolutionary technology developments have enabled vast amounts of biological information to be obtained, but from a medical standpoint it has yet to live up to the promise of associating individual genotypes to phenotypic states of wide-spread clinical relevance. The mechanisms by which complex phenotypes arise have been difficult to ascertain and the value of short-read sequencing platforms have been limited in this regard. It has become evident that resolving the full spectrum of genetic heterogeneity requires accurate long range information of individual haplotypes to be distinguished. Long-range haplotyping information can be obtained experimentally by long-read sequencing platforms or through linkage of short sequencing reads by means of a common barcode. This thesis explores these solutions, primarily through the development of novel technologies to phase short sequences of single molecules using DNA barcoding. A new method for high-throughput phasing of single DNA molecules, achieved by the production and utilization of uniquely barcoded beads in emulsion droplets, is described in Paper I. The results confirm that complex libraries of beads featuring mutually exclusive barcodes can be generated through clonal PCR amplification, and that these beads can be used to phase variations of the 16s rRNA gene which reduces the ambiguity of classifying bacterial species for metagenomics. Paper II describes a second methodology (‘Droplet Barcode Sequencing’) which simplifies the concept of barcoding DNA fragments by omitting the need for beads and instead relying on clonal amplification of single barcoding oligonucleotides. This study also increases the amount of information that can be linked, which is showcased by phasing all exons of the HLA-A gene and successfully resolving all the alleles present in a sample pool of eight individuals. Paper III expands on this work and explores the use of a single molecule sequencing platform to provide full-length sequencing coverage of six genes of the HLA family. The results show that while genes shorter than 10 kb can be resolved with a high degree of accuracy, compensating for a relatively high error rate by means of increased coverage can be challenging for larger genomic loci. Finally, Paper IV introduces the use of barcode-linked reads on an unprecedented scale, with a new assay that enables low-cost haplotyping of whole genomes without the need for predetermined capture sequences. This technology is utilized to generate a haplotype-resolved human genome, call large-scale structural variants and perform reference-free assembly of bacterial and human genomes. At a cost of only $19 USD per sample, this technology makes the benefits of long-range haplotyping available to the vast majority of laboratories which currently rely solely on short-read sequencing platforms.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. p. 45
Series
TRITA-CBH-FOU ; 2018:41
Keywords
Single molecule sequencing, DNA barcoding, whole genome haplotyping, linked-read sequencing, phasing, de novo genome assembly.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-235187 (URN)978-91-7729-939-4 (ISBN)
Public defence
2018-10-19, Air & Fire Auditorium, Tomtebodavägen 23, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20180919

Available from: 2018-09-19 Created: 2018-09-19 Last updated: 2018-09-19Bibliographically approved

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