Abstract
Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (>100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.