OCTOPUS: A simple, low-cost plasmid sequencing method

At Octant, we’re passionate about using the latest advances in synthetic biology to more rationally design drugs. In pursuit of our mission we developed OCTOPUS, a light-weight, cost-effective, and robust method for full-plasmid sequence verification using next-generation sequencing. OCTOPUS has accelerated much of Octant’s work over the last six months, and we think it will be broadly useful to the burgeoning community of startups and academics working to engineer biology.

Next-generation sequencing has fundamentally changed nearly every aspect of biological research. However, for the average scientist, it has yet to change the process of molecular cloning and sequence verification - a fundamental step in most molecular and synthetic biology R&D. Without access to expensive equipment and specialized processes, most cloning and sequence verification still uses outsourced Sanger Sequencing - a 60 year old technology that, while convenient at times, is quite painful at scale.

At Octant, we use a pooled cloning strategy that can generate hundreds of unique clones in a single reaction. Last year, as we scaled our cloning, we quickly outgrew our sequencing provider’s guaranteed turn-around time. They could commit to 192 individual reads per business day, which meant if we wanted to sequence 1000 colonies with four reads each, we could be waiting weeks for results. There were other limits to the existing Sanger-based process as well. For example, manually analyzing the data was about as painful as waiting for the results. In addition, we had to limit ourselves to only sequencing the parts of the plasmid we were changing. This is not ideal because it doesn’t find errors and mutations elsewhere in the plasmid, which when cloning at this scale, happen more than one would think. It became clear that our needs had outgrown the traditional approach. We needed a robust, scalable, and low touch method for verifying full plasmid sequences - ideally one that would not require a huge investment in labor and capital.

Around this time, we learned about Riptide, a new bacterial genome sequencing kit from iGenomX. In experimenting with Riptide we discovered that it works with crude lysate as the input. This combination is ideal for plasmid sequencing because it obviates the need for labor-intensive plasmid purification. We then built a turn-key computational pipeline to analyze the resulting data for plasmid verification - and thus OCTOPUS was born. OCTOPUS does not require specialized equipment or expensive liquid handling instruments, and the pipeline works on any modern desktop. All of this enables a single researcher to sequence over a thousand plasmids in less than a week.

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Here’s how OCTOPUS works in broad strokes:

  1. First, we pick colonies into normal 96-well plates and grow them overnight. Because we usually need 400-1200 colonies, it’s still easier and faster to do this manually than by colony picker. Also, colony pickers typically cost >$100K and don’t seem appropriate for our scale. 

  2. We then take a small aliquot of the cultures, gently lyse the cells to preferentially release plasmid over genomic DNA, and save the remainder as glycerol stocks.

  3. Next, we input the crude lysate directly into the iGenomX Riptide kit at 1/2x scale. The kit provides even coverage of our plasmids, works in lysate, does not require low-volume acoustic handling, and is cost-effective. iGenomX has also been super supportive, responsive, and helpful in the development of OCTOPUS.

  4. Then, we sequence on a Miseq. We’ve found that sequencing 384 colonies with a V2 Micro MiSeq kit seems to be the sweet spot in terms of price per well and turn around time. If you want to scale up, we run three 384 well plates on a standard V2 kit. You could also sequence 96 wells with a nano kit, but the price per well will go up.

  5. We then feed the sequencing data to our analysis pipeline, which automatically performs a number of quality control and processing steps to analyze what’s in each well.

  6. We’re now ready to pick clones for plasmid purification. Since our pipeline provides unique plate-well identifiers, it’s simple to integrate the output into your liquid handler of choice for downstream processing (we use the OpenTron’s OT2). 

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You can find details of the full protocol and the software on our GitHub. We hope people will use and extend OCTOPUS to fit their own needs, share their improvements with the community, and provide feedback. More broadly, we hope that others, especially the hundreds of new startups in synthetic biology, consider sharing pre-competitive ideas, methods, tools, etc. We think the community will be stronger as a result.

OCTOPUS is the result of practicing a core value of Octant’s scientific culture - a tight integration between wet and digital experimentation enhances both. For example, we add molecular barcodes to the backbone of our plasmids (an optional feature of OCTOPUS), which greatly simplifies the computational detection of contaminants. Conversely, this feature makes the analysis pipeline robust to dirtier data, allowing the bench to take experimental shortcuts. Everything we do at Octant depends on this type of practical convergence, which leads to dramatic increases in productivity. If this kind of interplay is something that you geek out on, please consider joining us.

Posted by

Henry Chan

Scientist

Nate Lubock

Scientist

Octant Moves to Emeryville!

Octant started about two years ago with the mission to use synthetic biology to map the space between chemicals and drug receptors to build better drugs. I joined in the first few weeks, working out of a café, to build and launch our first lab at the UCLA Magnify incubator space. There, we pressed forward to build a working prototype of our platform and plan a bigger future for drug discovery.

Last summer we made the difficult decision to leave the sunny beaches and taco trucks of LA for the Bay Area. The diverse biotech scene in the Bay Area felt like a better fit as we build a company bridging the gaps between synthetic biology, drug discovery, and large scale bio-computation. This is the story of how we uprooted our first lab (and all its members) and moved to a permanent home in record (or so we’re told) time.

'Inch wide-mile deep' is basically a PhD. As a newly minted PhD, I was an expert on how mammalian cells communicate following wounding. Unfortunately, not many employers were looking for a cell communication expert. But, fortunately, the “soft skills” I acquired as the first student in a new academic lab came in more handy that I could have imagined.

I had the unique experience of moving a lab not once, but twice during my PhD. One would think that after two of these experiences, I’d be an expert (or maybe refuse to do it a third), but as it turns out, having the backup of a well-funded university with template lab designs, monolithic purchasing power, and facilities staffed by building managers, custodial staff, and administrators made moving an academic lab, in retrospect, look easy.

Moving Octant, we were on our own. We started looking in October. Our lease at Magnify would be up the next August, and some of us needed to move families before then. We restricted our search to functioning lab spaces, having been warned that it would realistically take at least 9 months to build our own. Three months later, as I was wrapping up my maternity leave, and after several busted once-promising leads, we still had not found a single functioning lab space for Octant. With summer just a few months away, we were nearing panic mode. Our only options in hand were a sublet that we couldn’t confirm would even be available until late March and a lead on an old printing warehouse in Emeryville with ‘great bones’ and a future-thinking owner, but that would require a significant build.

We decided to go for the ‘build’ option. We mobilized a core team of a real estate agent, builder, architect, and several consultants to brute force in parallel over four months what we were told would take at least nine. No options were off the table, and we reexamined every assumption in the typical bio-lab construction process. Did our lab really need a plumbed gas line? Suction? Where do we absolutely need sinks? Where is AC necessary? Can we repurpose the building’s existing ductwork? Can the roof handle a big enough air handler? Is there a solution that avoids the need for structural review? We pushed the build team to get creative with a hard end-of-summer move-in deadline, while the rest of the team hit Zillow to find a place to live (and some SOs started looking for new jobs).

There were a variety of mishaps and lessons learned along the way! For example, the time our casework installer disappeared on an off-grid cross country bike ride for 2 weeks. Or when I interpreted 'outlet' to mean one socket (it meant two) and we nearly fainted at electrician costs that were double what we expected (go figure). Or when our freezers arrived on the generator truck, and we discovered that our new 220V outlets were configured incorrectly. We’re even still waiting on a replacement for a lost luminometer cable, which is threatening the timeline on a few experiments.

There are also a bunch of seemingly mundane day-to-day operations we took for granted at the incubator. Tasks like: tracking down packages without a receiving department, figuring out where to put our dumpsters on our crowded street, last-minute re-ups of our liquid nitrogen during a heat wave, negotiating with vendors absent much purchasing power, and our custodial staff (and sometimes our teammates) setting off our newly installed security alarm system at random times of the night.

But we did it. And we love our new space! Although the Magnify incubator was great and came with a lot of perks, we were bursting at the seams. Now we have a beautiful office, complete with individual standing desks, designated areas for science discussions, conference rooms, and data-inspired art on the walls. We have 4 lab spaces—Molecular Biology for our core synthetic biology work, Tissue Culture to engineer our mammalian cell libraries, Chemistry for drugs to test on our screening platform, and Sequencing to house our Illumina DNA sequencers. Plus, we have ample storage room, keeping the lab spaces clutter free and easy to work in.

Sure there were some challenges, but coming from a cramped office and an overcrowded shared lab space, we're definitely enjoying the extra leg room and opportunity to make everything our own. And most of all, we’re excited to be even further on our journey towards mapping a new frontier of biology to help treat human disease.

Posted by

 

Naomi

Scientist