Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules

Nucleic Acids Research
David RedinAfshin Ahmadian

Abstract

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 molecule...Continue Reading

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Citations

Nov 22, 2017·Nucleic Acids Research·Vijay KumarDan Levy
Dec 4, 2019·Scientific Reports·David RedinAfshin Ahmadian
Mar 6, 2018·Current Opinion in Structural Biology·Kritika Gupta, Raghavan Varadarajan
Oct 15, 2019·Bioconjugate Chemistry·Christiane StillerAmelie Eriksson Karlström

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Datasets Mentioned

BETA
SRP100498

Methods Mentioned

BETA
genotyping
Droplet Barcode Sequencing
DB-Seq
PCR
Assay

Software Mentioned

Picard
bowtie2
DB
Seq

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