Exploring the hepatitis C virus genome using single molecule real-time sequencing

Figure 1 Comparison of sequencing platforms. A: Direct sequencing (Sanger sequencing). This conventional sequencing method determines the consensus sequence of target regions. Nucleotide variants with allele frequencies of approximately 15% can be detected; B: Targeted deep sequencing using conventional short-read next-generation sequencing (NGS) can detect low abundance variants making up approximately 1% of total mapped reads; C: When long PCR products are used as templates for conventional short-read NGS, they are first fragmented into 100-200 bp segments, ligated to sequence adapters, amplified and then sequenced. The sequenced reads are mapped to a reference sequence using the shotgun method. One of the limitations of this technique is a lack of information regarding whether two distant mutations co-exist on a single template molecule; D: Third-generation sequencing methods represented by single-molecular real-time sequencing can generate ultra-long reads of more than 10000 bp, and contiguous sequence information can be obtained. NGS: Next-generation sequencing.