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Barcoded DNA Sequencing for Parallel Protein Detection
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis.

In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II).

As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events.

Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1.

Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , vii, 67 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:4
Keyword [en]
DNA barcoding, antibody labeling, antibody oligonucleotide bio-conjugation, DNAassisted proteomics, immuno-sequencing (I-Seq), droplet-based system, large-scale data analysis
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-159506ISBN: 978-91-7595-433-2 (print)OAI: oai:DiVA.org:kth-159506DiVA: diva2:785375
Public defence
2015-02-27, Air and Fire Lecture Room, SciLifeLab, Tomtebodavägen 23A, Solna, Stockholm, 14:15 (English)
Opponent
Supervisors
Note

QC 20150203

Available from: 2015-02-03 Created: 2015-02-02 Last updated: 2015-02-03Bibliographically approved
List of papers
1. Magnetic bead assisted labeling of antibodies at nanogram scale
Open this publication in new window or tab >>Magnetic bead assisted labeling of antibodies at nanogram scale
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2014 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 1, 14-18 p.Article in journal (Refereed) Published
Abstract [en]

There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

Keyword
Antibody, Automated protocol, Biotinylation, Labeling, Nanogram scale, Technology
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-142519 (URN)10.1002/pmic.201300283 (DOI)000330727000003 ()2-s2.0-84892410462 (Scopus ID)
Funder
VinnovaKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140306

Available from: 2014-03-06 Created: 2014-03-06 Last updated: 2017-12-05Bibliographically approved
2. Parallel barcoding of antibodies for DNA-assisted proteomics
Open this publication in new window or tab >>Parallel barcoding of antibodies for DNA-assisted proteomics
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2014 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 21-22, 2432-2436 p.Article in journal (Refereed) Published
Abstract [en]

DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

Keyword
Antibody, Bio-conjugation, DNA-assisted proteomics, DNA-barcoding, Massively parallel sequencing, Technology
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-157615 (URN)10.1002/pmic.201400215 (DOI)000344807600007 ()25263329 (PubMedID)2-s2.0-84909579700 (Scopus ID)
Funder
VinnovaKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20141212

Available from: 2014-12-12 Created: 2014-12-11 Last updated: 2017-12-05Bibliographically approved
3. Droplet-based Immuno-Sequencing to Deconvolute Affinity Recognition Events
Open this publication in new window or tab >>Droplet-based Immuno-Sequencing to Deconvolute Affinity Recognition Events
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-159513 (URN)
Note

QS 2015

Available from: 2015-02-03 Created: 2015-02-03 Last updated: 2015-02-03Bibliographically approved
4. Massively Parallel MHC-Typing by Sequencing RevealedNovel Variants of Canine Leukocyte Antigen
Open this publication in new window or tab >>Massively Parallel MHC-Typing by Sequencing RevealedNovel Variants of Canine Leukocyte Antigen
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-159515 (URN)
Note

QS 2015

Available from: 2015-02-03 Created: 2015-02-03 Last updated: 2015-02-03Bibliographically approved

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