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Single molecule DNA barcoding
KTH, School of Biotechnology (BIO).
2013 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Massive parallel sequencing (MPS) techniques provide huge opportunities in the area of life sciences. During the last five years, high-throughput sequencing platforms have become readily accessible. However, these platforms are expensive and the race to develop platforms at a reduced cost is still open. Netiher researchers nor practitioners have been able to reach a point at which routine sequencing (not including bacterial genomes) of high numbers of genomes is possible, and consequently it is often desired to select genomic regions of interest and enrich these regions before sequencing. In order to save money, barcoding of DNA has become popular during the last years. A traditional approach for using barcodes is to synthesize one barcode per sample and run all reactions in parallel and pool them toether before amplicification or sequencing. This traditional approach is very costly and is often nos used for batches larger than 100 samples.

To create a library of barcodes that is cheap, one oligo can be synthesi<ed with degenerated bases (N). If 20 random degenerated bases are used in this way, this means that 420 = 1.1 trillion combinations of barcodes are available. In practice, this implies that millions of barcodes are made for the same coast as that for one with the traditional approach. All the barcodes will be mixed in one tube and a physical separation of the barcodes can be accomplished by doing emPCR. We thereby perform compartmentalization and clonal amplification barcode on its surface (93%) by using a copy per bead (cpb) of 0.08. This result indicates that we successfully performed compartmentalization and monoclonal amplification of the barcodes on the bead surface.

Through the arrival of massive parallel short-read sequencing tools, the cost of sequencing DNA has been reduced significantly during the last years, thereby making it possible to sequence hundreds of bacertial genomes in one run. Still, the reduced length of the reads, compared with capillary-based methods, forms novel challenges in genome assembly. We have designed a method where a Nextera library preparation of genomic DNA fragments is coupled to one monoclonally amplified barcode on a bead, thereby achieving single fragment resolution of the sequencing data.

Place, publisher, year, edition, pages
Keyword [en]
Single molecule barcoding sezuencing DNA
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-150572OAI: diva2:744730
Available from: 2015-04-17 Created: 2014-09-05 Last updated: 2015-09-22Bibliographically approved

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