There have been many changes in sequencing technology over the course of my scientific career. In one of the research labs I rotated in as a graduate student, I assisted a third-year grad student with a manual radioactive sequencing gel because, I was told, “every student should run at least one in their career”. My first job after graduate school was as a research assistant in a lab that sequenced bacterial genomes. While I was the one creating shotgun libraries for the DNA sequencing pipeline, the sequencing reaction was performed using dideoxynucleotides labeled with fluorescent dyes and amplified in thermal cyclers. The resulting fragments were separated by manual loading on tall slab polyacrylamide gels (Applied Biosystems ABI 377s) or, once the lab got them running, capillary electrophoresis of four 96-well plates at a time (ABI 3700s).
Sequencing throughput has only increased since I left the lab. This was accomplished by increasing well density in a plate and number of capillaries for use in capillary electrophoresis, but more importantly, with the advent of the short read, massively parallel next-generation sequencing method. The next-gen or NGS technique decreased the time needed to sequence because many sequences were determined at the same time, significantly accelerating sequencing capacity. Instruments have also decreased in size as well as the price per base pair, a measurement used when I was in the lab. The long-prophesized threshold of $1,000 per genome has arrived. And now, according to a recent tweet from a Nanopore conference, you can add a sequencing module to your mobile device:
Welcome to the future – DNA sequencing on your mobile phone – imagine where and how you can use it. Hats off to the @nanopore team for getting this to work at this form factor, voltage and watts. https://t.co/Tm6A5fj8M4
— Ewan Birney (@ewanbirney) November 30, 2017
But none of this technology to perform high-throughput rapid automated sequencing would be possible without Leroy Hood and the prototype automated DNA sequencer created in his laboratory. As part of the Wisconsin Science Festival and Wisconsin Book Festival, I had the opportunity to listen to Luke Timmerman and Dr. Lloyd M. Smith, Director of the Genome Center of Wisconsin and a former post-doctoral student in Leroy Hood’s lab, discuss “Hood: Trailblazer of the Genomics Age,” the biography of Leroy Hood written by Timmerman. Dr. Smith was not only a source used for Timmerman’s book, but he was one of four people that was named on the patent for an automated DNA sequencing instrument.
Timmerman talked about Leroy Hood from his childhood in Montana to his undergraduate education at CalTech. He mentioned Hood’s time in medical school at Johns Hopkins as well as his time served at the NIH Public Health Service before Dr. Hood headed back to CalTech to start his own lab.
Hood not only wanted to investigate big questions in science, but wondered how to improve available tools to investigate the questions. In 1980, there were a series of good experiments using manual radioactive sequencing from Sanger. Being the visionary he was, Hood said we need to automate DNA sequencing. If this process could be high speed and high volume, lots of data could be generated quickly—an insight that could usher in an unprecedented throughput in genomics.
When Smith joined Hood’s lab in 1982, he was one of 70 people there doing research. Because Smith had a biophysics and instrument background, he ended up involved in the work on a sequencing instrument. Not surprisingly, during the development of an automated sequencing instrument, there was tension over who had what idea. Mike Hunkapiller pressed for four dyes versus one dye. We think little of having four fluorescent dyes assigned to each base (A, T, G, C), but this concept added more technical challenges to an already large technical task. Smith explained it took four years to make four color base tag sequencing because no one know how to attach fluors to DNA.
Two of the four dyes came along well, Smith commented, but one in particular was harder to synthesize. Smith did manage to have four dyes working at one time, but one had a low signal. Hood did what a good mentor should do: Pushed back on the low signal dye. Thus, Smith did more engineering to get the chemistry to work well. The end result: Lloyd Smith was the first author on a paper describing automated DNA sequencing and listed on the patent for the automated DNA sequencing technique.
In June 1986, a prototype of the automated DNA sequencer was announced in a Nature publication. CalTech held a huge press conference to announce this new machine. Leroy Hood may have been the visionary that drove others to fulfill his vision, but when making his remarks about this prototype instrument, Hood did not mention any of the others who worked for years getting the DNA sequencer to work. As Timmerman explained, not naming those who deserve credit eroded Hood’s standing both in his lab and the CalTech campus. However, the breakthrough in automated DNA sequencing drove the development of Applied Biosystems Incorporated to bring the instrument to market, offering a potential tool for sequencing the human genome.
The genomics era would not have come to fruition without Leroy Hood and those in his lab who labored to make an automated DNA sequencer reality. From slab gels to capillary electrophoresis that were used for sequencing during the Human Genome project to the immense throughput of NGS and sequencing an entire genome in hours, automated sequencing of DNA has become integral to genomics research. Personalized medicine is being practiced today because we have four fluorescent dyes that can be used to indicate the DNA sequence and machines that can rapidly detect those dyes, translating color to sequence. The genomics age is filled with possibilities with the use of four dyes to illuminate A, T, G and C and sequencing instruments that are as mobile as our personal devices.
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