Schematic representation of the experimental design.

<p>(A) Individual viral sequences within a quasispecies are assigned with a unique tag. In this example, three viral sequences are present in the quasispecies. Horizontal black lines represent individual viral sequences. Red circle represents mutation from the consensus. Colored tag represents unique tag assigned to individual viral sequences. The same tag is distributed to every sequencing reads originated from the same viral sequence. During quasispecies reconstruction, the tag contains the phasing information to reconstruct individual viral sequences. (B) A cassette consisting of a constant region (Constant), a restriction site (R1), a random oligonucleotide (tag) and the forward Illumina adapter (5′ FlowCell) is added to the 5′ end of the DNA sample. Individual DNA molecules in the resultant pool will each be afforded with a unique tag. (C) The input pool in this PCR step contains a limited number of DNA templates to reduce the complexity of the pool. The resultant PCR amplification generates multiple copies of individually tagged DNA templates. (D) The DNA pool is then divided into a series of PCRs with a second restriction site (R2) added to the 3′ end of the reaction product. (E) Ligation with population ID, which is a short specific DNA sequence serving as a barcode for multiplex sequencing, utilizes the two restriction sites, R1 and R2. (F) Amplicons with similar size are generated from different ligated DNA pools. Reverse Illumina adapter (3′ FlowCell) is added. Different amplicon pools can then be mixed and subjected to Illumina sequencing.