Author / Benjamin Gowen

A few weeks ago, Jacob wrote a blog post about his recent experience with posting pre-prints to bioRxiv. His verdict? “…preprints are still an experiment rather than a resounding success.” That sounds about right to me. I’m bearish on pre-prints right now because the very word implies that the “real” product will be the one that eventually appears “in print”. Don’t get me wrong--I think posting pre-prints is a great step toward more openness in biology, and I applaud the people who post their research to pre-print servers. Pre-prints are also a nice work-around to the increasingly long time between a manuscript’s submission and its final acceptance in traditional journals; posting a pre-print allows important results to be shared more quickly. There’s a lot of room for improvement, though. With some changes, I think pre-print servers could better encourage a real conversation between a manuscript’s authors and readers. Here are some of my thoughts on how they might achieve that. I know there are several flavors of pre-print servers out there, but for this post I’m going to use bioRxiv for my examples.

Improve readability

It’s 2016, we’ve got undergraduates doing gene editing, but most scientific publications are still optimized for reading on an 8.5x11” piece of paper. Pre-prints tend to be even less readable--figures at the end of the document, with legends on a separate page. The format discourages casual browsing of pre-prints, and it ensures the pre-print will be ignored as soon as a nicely typeset version is available elsewhere. I will buy a nice dinner for anyone who can make pre-prints display like a published article viewed with eLife Lens.

Better editability

bioRxiv allows revised articles to be posted prior to publication in a journal, but I would like a format that makes it really easy for authors to improve their articles. Wikipedia is a great model for how this could work. On Wikipedia, the talk page allows readers and authors to discuss ways to improve an article. The history of edits to a page shows how an article evolves over time and can give authors credit for addressing issues raised by their peers. Maintaining good version history prevents authors from posting shoddy work, fixing it later, and claiming priority based on when the original, incomplete version of the article was posted.

Crowd-source peer review

Anyone filling in a reCAPTCHA to prove they’re not a robot could be helping improve Google Maps or digitize a book. What if pre-print servers asked users questions aimed at improving an article? Is this figure well-labeled? Does this experiment have all of the necessary controls? What statistical test is appropriate for this experiment? With data from many readers about very specific pieces of an article, authors could see a list of what their audience wants. It looks like we need to repeat the experiments in Figure 2 with additional controls. Everybody likes the experiments in Figure 3, but they hate the way the data are presented.

Become the version of record

Okay, this one’s a definitely a stretch goal. Right now pre-prints get superseded by the “print” version of the article, but that doesn’t need to be the case. Let’s imagine a rosy future in which articles on bioRxiv are kept completely up-to-date. Articles are typeset through Lens, making them more readable than a journal’s PDF. There’s a thriving “talk” page where readers can post comments or criticisms. Maybe the authors do a new experiment to address readers’ comments, and it’s far easier to update the bioRxiv article than to change the journal version. At that point, bioRxiv would become the best place to browse the latest research or make a deep dive into the literature. Traditional journals could still post their own versions of articles, provided they properly cite the original work, of course.

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For the last couple of months, a few of us at the IGI have been generating new sgRNA libraries for CRISPRi and CRISPRa. After scraping colonies off of nearly one hundred extra-large LB-Agar plates, it was time to fill the lab with the sweet smell of lysed bacteria and DNA prep buffers. We were working with 21 separate sublibraries, totaling around 250,000 sgRNAs. Plasmid prep on this scale is a completely different beast from anything I had done before, so we decided to share some thoughts on what works (and what doesn't!) for efficiently prepping sgRNA libraries.

Prepping the work station

We were worried about other plasmids sneaking into our preps--especially individual sgRNA plasmids that get used frequently in our lab. We doused and scrubbed our benches and vacuum manifold with 70 % ethanol and RNase-Away before starting, and a few times throughout the day. This should hopefully destroy or denature any stray plasmids hanging around. It's also worth cleaning out your vacuum trap and putting fresh filters in the vacuum line, since old dirty filters can really weaken vacuum power.

Do all the DNA prep at once

For me, it's much more efficient to spend a couple of days solely devoted to high-throughput DNA prep than to spread the work out over several days, a few columns at a time. 

Teamwork

The initial lysis and neutralization steps in most plasmid preps are time-sensitive, so there's a limit on how many samples one person can process at once. We found that a team of 3 people (each processing 8 samples at once) maximized our throughput without us bumping into each other too much. After eluting DNA off the columns, once person can manage the DNA precipitation while others start on the next round of samples.

Starting material

Scraping the colonies off of a 23x23 cm LB-Agar plate gave us an average bacterial pellet mass of 1.1 g (range 0.5-1.6 g). This meant that each plate of bugs got its own maxiprep column (see below for kit recommendations). If you're working with bugs from liquid culture or other plate sizes, you can pool or aliquot the samples to get a similar pellet mass per column.

Plasmid prep kits

We wound up trying several different plasmid prep kits, and the clear winner in our hands was the Sigma GenElute HP Plasmid Maxiprep Kit. The columns are compatible with the QIAGEN 24-port vacuum manifold we already had in the lab, the protocol was amenable to doing 24 preps in a batch, and the house vacuum system in our building was strong enough to pull liquid through all 24 columns at once. Importantly, all of the columns ran consistently and reasonably quickly. One slow or plugged column is an annoying but solvable problem when doing 4 or 5 preps, but it can really back up the pipeline when doing multiple batches of 24. Our  average yield from this kit was 1.4 mg per prep.

Kits to avoid:

• Sigma GenElute HP Plasmid Megaprep: Sigma advertises 4 times the yield from a megaprep column compared to their maxipreps. Some of our samples could be pooled, so we thought pooling 4 samples into one megaprep would be faster than running them as 4 individual maxipreps. Boy were we wrong! The megapreps had to be processed one or two at a time, and thus didn't scale well at all. Worst of all, the megaprep columns were NOT compatible with the QIAGEN vacuum manifold. We managed to fix this with tubing and adapters, but the house vacuum system was only strong enough to pull on one or two of the larger megaprep columns at a time. For us, mega preps took far more time and gave about half the yield we would have expected from just grinding through 4 times as many maxipreps.
• QIAGEN Plasmid Plus Maxiprep Kit: 1 out of the 8 columns we used  stalled while running the cleared lysate. That column had to be left on the vacuum overnight. Our yields were also lower than the Sigma maxipreps. 
• QIAGEN HiSpeed Plasmid Maxiprep Kit: These don't scale well at all. The columns aren't compatible with a vacuum manifold, and the QIAprecipitator syringe filters require a lot of manipulations to each individual sample. After the first 4 samples, I ditched the QIAprecipitator step altogether. Precipitating the DNA with a 45 minute spin was much faster when dealing with 10 or 20 preps at once.

We're always interested in ways to make the next sgRNA library prep easier than the last. If you have your own favorite plasmid prep kit or other tricks for efficient library preparation, feel free to leave a comment. 

Special thanks to the other members of Team DNA Prep--Gemma Curie, Amos Liang, and Emily Lingeman. I'd still be running maxipreps if it weren't for them!

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