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Figure 1 | Genome Biology

Figure 1

From: BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads

Figure 1

Outline of BAsE-Seq methodology. (a) The goal of library preparation is to attach unique barcodes to full-length HBV genomes, and then juxtapose the assigned barcode to random overlapping fragments of the viral genome. A unique barcode is first assigned to each HBV genome using PCR. The two barcode assignment primers contain HBV-specific sequences on their 3′ ends, universal sequences (green) on their 5′ ends, and one of the primers also contains a random barcode (blue). Subsequently, barcode-tagged genomes are clonally amplified by PCR using primers that anneal to Uni-A and Uni-B and that add a biotin label (Bio) to the barcode-proximal end. The barcode-distal end is digested with exonuclease to obtain a broad size distribution of nested deletion fragments. Barcode-containing fragments are purified using Dynabeads, and intramolecular ligation of these fragments yields a library of circular molecules in which different regions of each HBV genome are juxtaposed to its assigned barcode. The circularized molecules are used as a template for random fragmentation and adapter tagging following the Nextera protocol. During PCR enrichment, a set of primers is used to incorporate Illumina-specific paired-end adapters and enrich for barcode-tagged molecules during sequencing. (b) Bioinformatics workflow. Barcode-containing read pairs are used to obtain a 'bulk consensus' genome by iterative alignment of read pairs against a GenBank sequence. Aligned read pairs are de-multiplexed into individual genomes based on barcode identity. Consensus base calls are extracted to obtain 'individual consensus' genomes and SNVs are identified in each genome to construct haplotypes.

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