Ms. Alicia Burt: Hello, and thank you for attending the Agilent webinar on Automated Next-Gen Sequencing Sample Preparation. My name is Alicia Burt, and I am a Senior Product Manager in Agilent Automation Solutions
Today, I would like to talk about an overview of the automated solutions that Agilent has developed for Next-Gen sequencing sample preparation. I will discuss the array of hardware that’s available for various Next-Gen sequencing applications, and I will show some references to the use of these products in the literature
The Agilent Next-Gen sequencing workstation is an open platform. We have developed automated methods for a variety of reagents and for a variety of sequencing platforms. And the basic principle is that using the Agilent Next-Gen sequencing Bravo, that you see in the central position of the photo on the left there, you can use virtually any commercially available reagent for almost all Next-Gen sequencing platforms. Today, we have automation protocols available for both Illumina and solid sequencers. We have an optimized protocol and user manual available for SureSelectXT reagents from Agilent for use on Illumina sequencers. Agilent has also developed automation protocols for SOLiD 4, SOLiD 5500, TruSeq RNA and DNA reagents and for NimbleGen library preparation and capture. We at Agilent are working together with our automation business as well as our Genomics business to continually evolve the reagents and also protocols available to support various reagents and sequencers
Here’s an overview of the tools that Agilent has developed for various stages of the Next-Gen sequencing process starting from the left in the orange with library prep all the way through to data analysis using our GeneSpring software. We have reagents with our SureSelect products and now through a recent acquisition the HaloPlex products for sample preparation. And we also do sample QC via the TapeStation and automation for library preparation from Agilent Automation Solutions, and last, but not the least, are the PCR instruments you see on the bottom of the page
So, before we get into the actual protocols that we have available, I think it dares mentioning the process for using magnetic bead separation to do purification. So, at the core of this process for Next-Gen sequencing library preparation there are a series of several purification steps. When you’re doing that by hand it can be quite labor intensive. And in this slide from one through six you can see the processes of doing the separation with the magnetic bead
So, starting in the beginning you’re binding the nucleic acid to the beads, exposing them to a magnetic. The beads are drawn to the magnetic, and then you can wash, and then elute the nucleic acid that you’re looking for. So, this process using those magnetic beads and the rings on the deck of our Bravo platform is the core for many of the separations that take place during automated sequencing sample preparation
So, on the next slide you see an overview of the library preparation portion of our protocols. Starting at the very top here you’re sheering genomic [sp] DNA, that’s the starting sample for most of the protocols, and working through quantitation quality check, preparing the ends, adaptor ligation, PCR enrichment and again another quality check. This process by hand has many, many labor intensive pipetting steps. Here you can see there are five steps for automated purification. In each of these steps the Bravo automated liquid handling platform is automating the magnetic bead separation and purification to add consistency and reliability to your results
So, for this step of library preparation on the deck of the Bravo takes about four hours. This process is what you do before whole genome sequencing. So, the Bravo will automate a whole genome sequencing library preparation for up to 96 samples in four hours. There are other steps of the process that are specific to targeted enrichment or targeted resequencing
So, if you’re doing SureSelect targeted enrichment you can see you have the prepped library, and then in the case for targeted resequencing you’ll use the SureSelect for targeted enrichment. Here you see which portions of that take place on the deck of the Bravo. We do the hybridization overnight. That takes place off deck on a Thermal Cycler. So, this is the next step of the process for hybridization. And then, on the next slide you see automated capture and wash. Again, this part is specific to targeted resequencing using SureSelect or other methods. And you see here the process starting with the captured product on the beads going through the wash steps, PCR enrichment and quantitation. Here you see which steps of that are taking place on the deck of the Bravo
The reason why I called this out is because it helps to know what is happening on the deck of the Bravo and what is happening on other instruments in the lab, for example, PCR instrument or a sample QC instrument
On the next slide you can see how you can process 192 targeted enrichment samples in one work week. In this color coded scheme using one color for yellow for the first plate and the second color blue for the second plate, you can see about how the workflow happens in a laboratory. Assuming that you have completed the sheering for both plates of samples the prior week, let’s say on Friday, you can come in Monday morning and initiate the library preparation for the first plate. And then, Tuesday morning you start the library process for the second plate. And by doing that one person can do two full plates a week processing 192 samples for targeted enrichment. If you’re doing whole genome sequencing it’s quite different because that process is much shorter. Instead of doing two plates a week you could actually do two plates a day. So, the throughput for whole genome sequencing targeted preparation is much faster
One other thing to mention is that the time investment in the lab to process the samples is one part is the liquid handling and another part is the sample QC, which you can see here in each process for each plate you’re doing sample QC three times. And so, by using a high throughput QC instrument you can actually save a lot of time in the process. I’ll give you a little bit more information on that later
Our quickest method for doing sample QC today is using the Agilent TapeStation, and processing 96 samples on a TapeStation takes approximately 1.5 hours. So, you do that three times per plate
This is a VWorks animation software. You can see in the upper right-hand corner there is a diagram of the Labware MiniHub. This is prompting the user to put the appropriate reagents and labware in the MiniHub. In the central part of the right side is the Bravo deck showing which accessary is on the Bravo and which labware should be on the Bravo
You can see the pull-down menus that the user uses to select the appropriate protocol to use and also the number of samples that are being processed. The number of samples is a very key feature in the software that tells the Bravo how to utilize the tips, how to load the tips for various numbers of samples
On the next slide I show two options for automation. The upper left is our basic configuration for initiating Next-Gen sequencing automated sample prep. That is what we call the Option A. It includes on the right side an automated liquid handling platform, the Bravo, and also on the left side the PlateLoc Thermal Microplate Sealer. On the bottom is taking that same system and adding additional automation to give you more walkaway time. In addition to the liquid handler and plate sealer, you also see the BenchCel Microplate Handling System and also on the right the Labware MiniHub. The throughput for both of these instruments is exactly the same because that is governed by the throughput of the Bravo. What you get with the Option B is you get additional walkaway time in the lab by adding the BenchCel Microplate Handler and also the Labware MiniHub. By adding these two devices, you save yourself many manual interventions in the lab. Part of the Next-Gen sequencing process is very pipetting intensive. There can be 50 to 60 pipetting steps in one protocol. The BenchCel system will load new pipette tips onto the deck of the Bravo
In our labs at Agilent, you can see in the yellow section of the chart on the right, we have estimated that a very adept technician in the lab can process between 20 to 40 targeted enrichment samples in one work week. That same technician using the Bravo can increase their throughput up to five fold. The same technician at Agilent is now processing two full 96 plates a week of targeted enrichment sample prep. And one really key distinction here is the numbers that I’m citing are for targeted enrichment samples. If you’re doing whole genome library preparation that number is scaled up significantly, and you can do 10 plates a week or 960 samples per week
So, I’d like to show some data that shows the output of our automated system and how it performs with respect to sequencing. In this slide you can see we’re comparing the performance of the same sample that’s been prepared in 12 different wells on a Bravo deck. And what’s plotted here is the number of reads for the different targeted basis that we’re sequencing. In every case almost 100 percent of the targeted samples were sequenced. It’s only when you get to the redepth [sp] of 20 reads where you start to see a slight variation in the samples. If it’s 10 or fewer reads you can see that in this sample set there are very minute changes in the sample preparation
Another metric that we look at to access the quality of automated sample preparation is the yield, comparing yield of manual preparation to automated preparation. What you see plotted here on the left, in red, is the yield of DNA from an automated sample preparation using the Bravo and on the right manual preparation. And the key point to note here is by using the automated sample preparation it tightens the CV or it lowers the variation. On the left with the automated sample prep we see a CV of approximately 11 percent. And on the right with manual preparation we see a CV that’s greater, about 18 percent. What we look at when we are using the system or qualifying a system for use with Next-Gen sequencing is number one is the variation smaller than you would see in manual and number two are you getting sufficient yield to proceed with sequencing
Another look at what the variation is in sample preparation or how to look at that with an automated system for sample preparation is we’re looking at the percentage of reads that are near what you’re looking for. This is for targeted enrichment, looking at specific areas of the genome that you are targeting or pulling down and which percentage of the reads were within 100 basis or 200 basis or 300 basis of the sequences that you’re pulling down. The key point to take away from this chart is that if you look across 12 different indexes or 12 different wells the sample preparation yields very similar results. And that’s what we’re looking for here is consistency in sample preparation
I’d like to shift gears now and move away from talking about the protocols and start talking about the hardware and the different configurations that are possible to customize these solutions for your laboratory. This is the continuum of hardware that Agilent has engineered specific for Next-Gen sequencing sample preparation. On the far left here you see the Option A, which is the Bravo and the PlateLoc Thermal Sealer. That’s the minimum configuration for automated sample prep. And as you proceed to the right of the slide you can see larger configurations for increasing walkaway time. In the middle of the slide is what we call the Option B that has the BenchCel Microplate Handler and the MiniHub, and on the far right is the BenchBot based system for the possibility of higher customization and additional components can be added to that system for more walkaway time or for unique implementations in your lab
So, here’s a little detail about what is available with the Option A and the Option B. Option A is the Bravo Liquid Handling Platform including a 96 LT liquid handling head. And we also offer as part of this package five accessories to outfit the Bravo deck specific for Next-Gen sequencing applications. There’s one cooling pad, two Peltier heaters, a shaker, a magnetic bead plate. And then, also in the Option A is the standalone PlateLoc. The Option B contains everything Option A has plus the addition of the BenchCel 4R and the Labware MiniHub.
This slide shows an example of what we can do for custom Next-Gen sequencing applications. In the picture in the upper right-hand corner you can see a CAD drawing that we’ve developed for a customer showing the BenchBot, which is the tall arm, which has a radial reach. That increases the number of instruments that can be integrated in a workstation configuration. The reach of the BenchBot is 270 degrees. So, we now have the opportunity to put many other devices online or integrated with that instrument
So, this has all of the capability that you see with an NGS Option B and adds to it an integrated PlateLoc Thermal Microplate Sealer as well as a PCR instrument. The benefit of this system is, one, you can do everything in Option B does, but also offers less manual interventions by having the PlateLoc and the PCR instrument integrated. We have produced approximately six of these systems worldwide for customers. This just calls out the different names of the various pieces of hardware, the PlateLoc, the Bravo, BenchCel 4R and the MiniHub
Now, as I mentioned, the Bravo is the central component of either of our Next-Gen sequencing platforms. A couple of things to note about the Bravo, it has a very compact design. The depth of it is compact enough to fit on virtually any laboratory bench. And the head, which is in the middle of the diagram, is swappable. Meaning it’s very, very easy to take off the 96 head and put in a different head. For example some customers use the 96 head for Next-Gen sequencing applications and then also purchase a 384 well head for use in preparing PCR plates, for example. There are no electronics below the deck of the Bravo, and it comes equipped with a gripper to move labware around to different positions of the deck
On the next slide you’ll see a top view picture of the numbered deck positions on the Bravo, and the text here calls out which positions we put to the different components. You can change the configuration of a Bravo deck to move accessories to different positions of the deck. In the case of Next-Gen sequencing we have optimized the deck locations for the protocols that we’ve written. It’s certainly possible to modify many of the accessories to different locations if you choose
Here’s another top down view of the Bravo deck, and you can see the different accessories that we use for the Next-Gen sequencing application. On the left and right, in the middle, that’s the C-Pack [sp]. We call those the Peltier heaters. In the central position is the Teleshake. That is very instrumental in all of the magnetic bead purifications that we’re doing in Next-Gen sequencing. As the Teleshake shakes the pipetting motion will move the liquid up and down so there is a uniform mixture of the bead slurry. In the bottom left there’s a picture of the ringed magnets that are useful for the magnetic bead separation. And finally in the bottom right there’s a picture of the chiller. There is an off deck heat exchanger to maintain the temperature of the chiller
The one thing to call out here on the next slide, the inserts at the position four and position six these are used to make sure that the Peltier heating transfers the heat very reliably to the samples in the labware on the top of the deck
On the next slide, I have an animation that shows the movement of this system in the Next-Gen sequencing application. First, you see at the right of the system the Labware MiniHub, which is a random access carrousel. And the BenchCel robot comes over, picks up a piece of labware and will now transfer that to the deck of the Bravo. The Bravo has grippers to move the labware about the deck. And the BenchCel is a labware storage device. So, it can deliver fresh tips to the base of the Bravo at the third position on the Bravo platform. The Bravo picks up fresh tips, picks up reagents. And now the Teleshake is manipulating the beads as they’re pipetted up and down. So, that shows the movement of many of the accessories on the platform to see how the Bravo is moving, the BenchCel’s moving and the MiniHub
I did mention earlier in the slide about timesaving or planning the week in the lab on how you can optimize the throughput of your laboratory. One of the new products that Agilent has introduced in the last year or so is the TapeStation 2200. This particular instrument can be used at three steps in the Next-Gen sequencing process, and the TapeStation can process 96 samples in 1.5 hours. This instrument is a huge time savings over previous instruments that were used like the Bioanalyzer. You can cut time from four and a half hours to now one and a half hours using the TapeStation
The TapeStation uses the ScreenTape that you see at the top. That’s the consumable product that’s used. And the way that the TapeStation ScreenTape works is that you only use as many columns as you need. So, for example if you’re only processing three samples in the TapeStation, you can come back to that ScreenTape and use it at a different time. There’s no need to prime the ScreenTape cartridge. And you can see on the bottom there the output of the ScreenTape software for sample QC
This is a picture of the labware that we recommend for Next-Gen sequencing applications. The Bravo is compatible with any SBS formatted labware. The labware that you see here we have used in all the protocols that we’ve developed here. It’s certainly possible to use any labware that’s common in your laboratory or your institution. By using the labware that we recommend, you can get up and running faster. So, in many cases we get labs up and running within three days, because if they’re using our labware, we don’t have to reset the labware definitions in the software. If you have PCR plates that you prefer, you can just change the height settings and the gripper settings for the labware that you’re using. You can purchase these from--I have the names of the companies that provide them here and these are recommendations, but, again, you can use any that you like
I’d like to talk about some of the references where you can see the use of the Agilent NGS Bravo for extremely high throughput sample preparation. This was published in GenomeBiology early last year by the first author Sheila Fisher. In this paper they give a lot of recommendations for how to scale the throughput of Next-Gen sequencing sample preparation using the Agilent Bravo. In this paper they cited that they could process 1,200 targeted enrichment samples in a single week
This is a recent paper that I actually just found published in May of 2012 and--by Rohland and Reich in Genome Research. The goal of this work was to optimize or reduce the cost for sample preparation for Next-Gen sequencing. In this particular case the estimate of these researchers was the cost of the capital equipment, which included the Bravo, the Covaris and a PCR instrument. Using the financial accounting rules of their institution equated to approximately three dollars per sample. Also in this paper, they are now up to processing 2,000 samples per week. So, the point I’d like to make with both of these is that the Bravo has been the instrument of choice for labs that are scaling up to 2,000 samples a week, and number two by using the Bravo you get not only consistent results but a very cost effective solution for scaling the throughput
Agilent has developed a very comprehensive solution. We have both automation and the reagents developed and supported by a single company. In addition to SureSelect, we’ll be working on HaloPlex automation protocols, and we already have developed a number of protocols for other reagents that are available today
Agilent brings automation expertise through our acquisition of Velocity11. Agilent now has been automating life science workflows for over 12 years. We have a team available worldwide for both technical support and engineering support. We offer you flexibility in instrumentation. It’s not just a single box. As you saw before, you can scale the size of the instrumentation for your specific need, whether it’s just a Bravo or a Bravo plus plate handling or a customized BenchBot solution. We have satisfied customers, and we have over a hundred NGS systems in use worldwide. We are used by the key sequencing centers at the Broad, BGI and Sanger. They’ve all standardized using an NGS Bravo. And the two publications, which I showed, using the Bravo to scale throughput of sample preparation to up to 2,000 samples a week
If you would like more information on any of our automated sequencing sample preparation methods this is a link to the page on our website
Thank you very much for attending the webinar