Principle and design of molecular barcodes tagged on the viral genome
The molecular barcoding strategy has been successfully employed in “omics” techniques for virus studies (summarized in Table 1). The principle of molecular barcoding is to tag the viral genome with a string of random nucleotides in order to distinguish one virus from another. Molecular barcodes can be introduced into a template by using different approaches. First, molecular barcodes can be embedded into sequencing adaptors while constructing sequencing libraries (Figure 1A). A classical example was given by Schmitt et al. (2012)[19]. They constructed sequencing libraries with two independent molecular barcodes associated at both ends of each library to correct sequencing errors present on every sequencing read. It is noteworthy to point out the difference between “molecular barcodes” and “sample barcodes (sample indexes)” here. Although both are common to be used in the same sequencing reads, molecular barcodes aim to correct sequencing errors and increase sequencing accuracy or define true mutations; whereas sample barcodes enable multiple samples to be sequenced together, so called multiplexed into the same lane of flow cells (Figure 1B). The second approach is to employ molecular inversion probes (MIP) carrying molecular barcodes. A good example of this approach is the single molecule Molecular Inversion Probes method[23](Figure 1C), which combines the MIP strategy for targeted capture[32–34]at a single-molecule level[12,13,16,17,21–22]. Third, molecular barcodes can also be introduced on a template by PCR amplification with target-specific primers (Figure 1D).