Geospiza kicked off February by attending the AGBT and ABRF conferences. As part of our participation at ABRF, we presented a scenario, in our poster, where a core lab provides Next Generation Sequencing (NGS) transcriptome analysis services. This story shows how GeneSifter Lab and Analysis Edition’s capabilities overcome the challenges of implementing NGS in a core lab environment.
Like the last post, which covered our AGBT poster, the following poster map will guide the discussion.
As this poster overlaps the previous poster in terms providing information about RNA assays and analyzing the data, our main points below will focus on how GeneSifter Lab Edition solves challenges related to laboratory and business processes associated with setting up a new lab for NGS or bringing NGS into an existing microarray or Sanger sequencing lab.
Section 1 contains the abstract, an introduction to the core laboratory, and background information on different kinds of transcription profiling experiments.
The general challenge for a core lab lies in the need to run a business that offers a wide variety of scientific services for which samples (physical materials) are converted to data and information that have biological meaning. Different services often require different lab processes to produce different kinds of data. To facilitate and direct lab work, each service requires specialized information and instructions for samples that will be processed. Before work is started, the lab must review the samples and verify that the information has been correctly delivered. Samples are then routed through different procedures to prepare them for data collection. In the last steps, data are collected, reviewed, and the results are delivered back to clients. At the end of the day (typically monthly), orders are reviewed and invoices are prepared either directly or by updating accounting systems.
In the case of NGS, we are learning that the entire data collection and delivery process gets more complicated. When compared to Sanger sequencing, genotyping, or other assays that are run in 96-well formats, sample preparation is more complex. NGS requires that DNA libraries be prepared and different steps of the of process need to be measured and tracked in detail. Also, complicated bioinformatics workflows are needed to understand the data from both a quality control and biological meaning context. Moreover, NGS requires a substantial investment in information technology.
Section 2 walks through the ways in which GeneSifter Lab Edition helps to simplify the NGS laboratory operation.
In the first step, an order is placed. Screenshots show how GeneSifter can be configured for different services. Labs can define specialized form fields using a variety of user interface elements like check boxes, radio buttons, pull down menus, and text entry fields. Fields can be required or be optional and special rules such as ranges for values can be applied to individual fields within specific forms. Orders can also be configured to take files as attachments to track data, like gel images, about samples. To handle that special “for lab use only" information, fields in forms can be specified as laboratory use only. Such fields are hidden to the customers view and when the orders are processed they are filled later by lab personnel. The advantage of GeneSifter’s order system is that the pertinent information is captured electronically in the same system that will be used to track sample processing and organize data. Indecipherable paper forms are eliminated along with the problem of finding information scattered on multiple computers.
Web-forms do create a special kind of data entry challenge. Specifically, when there is a lot of information to enter for a lot samples, filling in numerous form fields on a web-page can be a serious pain. GeneSifter solves this problem in two ways:
First, all forms can have “Easy Fill” controls that provide column highlighting (for fast tab-and-type data entry), auto fill downs, and auto fill downs with number increments so one can easily “copy” common items into all cells of a column, or increment an ending number to all values in a column. When these controls are combined with the “Range Selector,” a power web-based user interface makes it easy to enter large numbers of values quickly in flexible ways.
Second, sometimes the data to be entered is already in an Excel spreadsheet. To solve this problem, each form contains a specialized Excel spreadsheet validator. The form can be downloaded as an Excel template and the rules, previously assigned to field when the form was created, are used to check data when they are uploaded. This process spots problems with data items and reports ten at upload time when they are easy to fix, rather than later when information is harder to find. This feature eliminates endless cycles of contacting clients to get the correct information.
Once order data are entered, the next step is to process orders. The middle of section 2 describes this process using an RNA-Seq assay as an example. Like other NGS assays, the RNA-Seq protocol has many steps involving RNA purification, fragmentation, random primed conversion into cDNA, and DNA library preparation of the resulting cDNA for sequencing. During the process, the lab needs to collect data on RNA and DNA concentration as well as determine the integrity of the molecules throughout the process. If a lab runs different kinds of assays they will have to manage multiple procedures that may have different requirements for ordering of steps and laboratory data that need to be collected.
By now it is probably not a surprise to learn that GeneSifter Lab Edition has a way to meet this challenge too. To start, workflows (lab procedures) can be created for any kind of process with any number of steps. The lab defines the number of steps and their order and which steps are required (like the order forms). Having the ability to mix required and optional steps in a workflow gives a lab the ultimate flexibility to support those “we always do it this way, except the times we don’t” situations. For each step the lab can also define whether or not any additional data needs to be collected along the way. Numbers, text, and attachments are all supported so you can have your Nanodrop and Bioanalyzer too.
Next, an important feature of GeneSifter workflows is that a sample can move from one workflow to another. This modular approach means that separate workflows can be created for RNA preparation, cDNA conversion, and sequencing library preparation. If a lab has multiple NGS platforms, or a combination of NGS and microarrays, they might find that a common RNA preparation procedure is used, but the processes diverge when the RNA is converted into forms for collecting data. For example, aliquots of the same RNA preparation may be assayed and compared on multiple platforms. In this case a common RNA preparation protocol is followed, but sub-samples are taken through different procedures, like a microarray and NGS assay, and their relationship to the “parent” sample must be tracked. This kind of scenario is easy to set up and execute in GeneSifter Lab Edition.
Finally, one of GeneSifter’s greatest advantages is that a customized system with all of the forms, fields, Excel import features, and modular workflows can be added by lab operators without any programming. Achieving similar levels of customization with traditional LIMS products takes months and years with initial and reoccurring costs of six or more figures.
The last step of the process is collecting the data, reviewing it, and making sequences and results available to clients. Multiple screenshots illustrate how this works in GeneSifter Lab Edition. For each kind of data collection platform, a “run” object is created. The run holds the information about reactions (the samples ready to run) and where they will be placed in the container that will be loaded into the data collection instrument. In this context, the container is used to describe 96 or 384-well plates, glass slides with divided areas called lanes, regions, chambers, or microarray chips. All of these formats are supported and in some cases specialized files (sample sheets, plate records) are created and loaded into instrument collection software to inform the instrument about sample placement and run conditions for individual samples.
During the run, samples are converted to data. This process, different for each kind of data collection platform, produce variable numbers and kinds of files that are organized in completely different ways. Using tools that work with GeneSifter, raw data and tracking information are entered into the database to simplify access to the data at a later time. The database also associates sample names and other information with data files, eliminating the need to rename files with complex tracking schemes. The last steps of the process involve reviewing quality information and deciding whether to release data to clients or repeat certain steps of the process. When data are released, each client receives an email directing them to their data.
The lab updates the orders and optionally creates invoices for services. GeneSifter Lab Edition can be used to manage those business functions as well. We’ll cover GeneSifter’s pricing and invoicing tools at some other time, be assured they are as complete as the other parts of the system.
NGS requires more than simple data delivery
Section 3 covers issues related to the computational infrastructure needed to support NGS and the data analysis aspects of the NGS workflow. In this scenario, our core lab also provides data analysis services to convert those multi-million read files into something that can be used to study biology. Much of this covered in the previous post, so it will not be repeated here.
I will summarize by making the final points that Geospiza’s GeneSifter products cover all aspects of setting up a lab for NGS. From sample preparation, to collecting data, to storing and distributing results, to running complex bioinformatics workflows and presenting information in ways to get scientifically meaningful results, a comprehensive solution is offered. GeneSifter products can be delivered as hosted solutions to lower costs. Our hosted, Software as a Service, solutions allow groups to start inexpensively and manage costs as the needs scale. More importantly, unlike in-house IT systems, which require significant planning and implementation time to remodel (or build) server rooms and install computers, GeneSifter products get you started as soon as you decide to sign up.