Why use PacBio's SMRT technology?

**Introduction**

The completion of the sequencing of the human genome in 2001 by the Human Genome Project marked the beginning of the genomic age. Though the human genome cost a billion(!) dollars, unbelievable decreases in the cost of DNA sequencing in the past decade have put full genome sequencing within reach for many organisms.

You may have even heard that a human genome can be sequenced for less than $1,000! This is nearly true, but it isn't the full story. In the case of human genomes, it costs around $1,000 dollars to "resequence" the genome, as the billion dollar Human reference genome is available to guide the process.

In the case of doing a totally new genome, like we propose for the Big Dipper firefly Photinus pyralis, it is a more expensive proposition. For sequencing the firefly genome, we decided to go with PacBio's SMRT DNA Sequencing technology, as it will produce the best quality genome that can serve as a reference for years to come.

**Why PacBio**

PacBio’s "Single Molecule Real Time", or SMRT, DNA sequencing technology produces long DNA “reads” (each read being the 10s of thousands of A,T,C, and G basepairs in a defined order), from across the firefly genome. In contrast, the short read sequencing sequencing technology used for the "$1000 human genome", while cheaper, only produces reads around 150-500bp long. Assembling DNA sequencing reads into a complete genome is a bit like assembling a giant jigsaw puzzle. By carefully fitting all the separate pieces together, you can finally reveal the bigger picture.

But imagine having the same jigsaw puzzle, but it comes it two sets: one made up with tiny puzzles pieces, the other with large pieces. This is analogous to assembling a genome with short-reads (the jigsaw set with the small pieces), or with long-reads. For the dedicated puzzler, the puzzle with large pieces is easier, but ultimately both the small piece and the large piece jigsaws can be completed, as each piece is unique or nearly so by design. But for the genomic jigsaw, things are more complicated. Genomes can be highly repetitive, containing things like "ATATAT..." repeated endlessly, or the genomic remains of long-dead and nigh-identical viruses that are spread throughout the genome. These repetitive regions can't be easily assembled, as they show up in multiple places in the genome, and when you obtain a DNA read from the middle of a repetitive region, even the most dedicated puzzler (or very powerful computer in the case of genome assembly), can't figure out where the piece truly goes. PacBio's big jigsaw pieces, the long-reads, ensures that more of these repetitive regions can be spanned by the read and thereby made "unique" as a repetitive region flanked by clearly unique regions, just as it appears in the true sequence of the genome. There are DNA sequencing tricks other than long-reads to get around the issue of repetitive regions in genomes, but with the continued decreases in cost and increases in read length from PacBio's SMRT technology, their technology has become the gold standard for sequencing new genomes.

Fireflies are real-world organisms. In contrast, many scientific studies focus on “model” organisms (like mice and fruit flies), where different individuals typically show high genetic similarity. In fact, scientifically inbred lines (which model organisms are) are literal genetic clones of one another! To deal with the real-world genetic heterozygosity of fireflies, PacBio’s long read sequencing will give us a huge advantage.

**Details on work thus far?**

<comment from SES> I would leave it here and do a second lab note with work so far.

<comment> Does Tim have any pics of him spooling DNA for PacBio?Pictures of raw data for DNA extraction / genome size measurement?