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Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules
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. Karolinska Institute (KI), Sweden.
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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2017 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 13, e125Article in journal (Refereed) Published
Abstract [en]

Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.

Place, publisher, year, edition, pages
Oxford University Press, 2017. Vol. 45, no 13, e125
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-212628DOI: 10.1093/nar/gkx436ISI: 000406776400008Scopus ID: 2-s2.0-85026371846OAI: oai:DiVA.org:kth-212628DiVA: diva2:1135724
Funder
Stiftelsen Olle Engkvist Byggmästare, 2015/347Knut and Alice Wallenberg Foundation, 2011.0113Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170824

Available from: 2017-08-24 Created: 2017-08-24 Last updated: 2017-08-24Bibliographically approved

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Redin, DavidBorgström, ErikHe, MengxiaoAghelpasand, HoomanKäller, MaxAhmadian, Afshin
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