Sample submission
Upon logging into the PIMS, users can immediately create a new sequencing order and see their last week's orders in the right top corner of the main PIMS page (Figure 1).
One of the frequent irritations of large computer systems is the difficulty of locating the desired information. Accordingly, the PIMS sequencing extension is designed to display only the information relevant to the user. For example, when the user logs into PIMS, the main page displays the orders recently submitted by this user. Older orders or orders which do not belong to the user are not displayed. Normally, this filtering is sufficient to allow the user to find the information they are interested in. Alternatively, the results can be located by searching with the unique sequence order number. It is also possible to search for individual samples by any of their details, e.g. template or primer names, principal investigator or user names and so on. All the information in PIMS is seamlessly linked and the list of results is paginated to ease navigation.
Creating a new sequencing order in PIMS is straightforward; just fill in the web form (Figure 2).
The required information can either be input via a web submission form (Figure 2) or by uploading a spreadsheet. The spreadsheet method allows the rapid submission of up to 96 samples at once, and is aimed for frequent facility users who know the rules, such as required concentrations and minimum sample volumes. Many fields come pre-loaded with the most frequently used values or allow selection of pre-defined values from drop-down lists. The PIMS sequencing extension validates the data, as it is important that the concentrations and volumes of templates and primers lie within predefined limits.
Once the requisite sample information has been submitted, the user is given a unique order identifier similar to the order number familiar to users from online shopping. Using the order number, users can always retrieve information on the samples in the order. The order can be amended after submission, for example, by removing, adding or editing samples, so long as a sequencing operator has not started processing it. Along with the order id, the users are given a list of the templates and primers, plus details of the volumes that should be provided to the sequencing facility. The list of primer (if any) and template names with minimal volume to be provided to the facility is submitted to the user email address together with the order confirmation. This email contains interactive links to the appropriate pages within the PIMS system allowing the users to review their order. The last stage of the sample submission process is the submission of the physical samples for sequencing. The samples must be labelled according to the names provided to the PIMS and the order number must be provided. Optionally the order confirmation email can be printed and supplied with the samples.
Sample processing
Since the sample submission forms are completed before the sample arrives at the facility, the facility operator knows in advance what to expect and so can plan accordingly. Sequencing operators have additional facilities in PIMS to help them with the sequencing process. The main page for the facility operator contains an overview of all plates in all processing stages. It immediately shows how many runs are being processed, how many samples remain to be processed, as well as how long they have been in the queue (Figure 3).
For the sequencing facility operator, the sample processing is a four stage process. Each processing stage represents a distinct operation in PIMS and can be performed at a time convenient for the operator. The four stages are:
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1)
New Order
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2)
Planned Run
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3)
Awaiting Results Run
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4)
Completed Run
where a "Run" represents a plate, or part of a plate containing different samples, possibly from different users' or customers' orders. The main page for the sequencing administrator reflects these steps.
Facility users submit their samples to the facility and record their sample details into the PIMS system effectively creating the new orders. From the sequencing facility operator point of view the new order stage is there their work begins.
The "Planned Run" is a plan, mapping user samples to the sequencing plate wells. The "Awaiting Results Run" is a sequencing plate for which the full chemical composition of the wells is known, such that the plate can be prepared by the liquid handling robot and consequently processed by the sequencer. The "Completed Run" - is a run which has been processed, i.e. samples sequenced and the sequencing results are uploaded into PIMS. In other words the completed run is where the facility operator hands the work he or she has done to the customers. A detailed explanation of each of these stages is provided below.
Sample processing operations are available for the sequencing operators only. Ordinary users do not have access to this functionality and thus are shielded from unnecessary details. At the same time, facility operators are in a full control of the sample processing and can be certain that no one can alter or indeed even view the data. Data protection is an important issue for any facility operating on samples from multiple users.
Planning the sequencing experiment
Planning of a sequencing experiment's functionality is only available to the sequencing operators. The sequencing operator plans the run from the sequencing administrator main page, which displays the list of orders to be processed, and calculates the total number of samples in single or multiple orders, as well as the number of plates required to sequence all the samples.
Samples need to be mixed with primers and other chemicals in a plate for sequencing. This can be done manually or using a liquid handling robot. The amount of sample in a well depends on its concentration and quality. To minimise the costs, the sequencing plate should have no or very few empty wells. At the same time, the users should not need to wait an excessively long time for their samples to be sequenced. Given the large number of samples processed by a typical sequencing facility, planning becomes a complicated process. In addition, one needs to keep track of all the samples on the sequencing plate to be able to match the sequencing results back to them. PIMS helps to meet these needs by proposing a plate plan automatically, taking into account all the variables mentioned above.
Once the planned run is recorded, any orders it contains can no longer be amended and are no longer displayed on the new orders lists. However, it is still possible to abandon the plan and start everything from scratch.
Manual or Robotic handling of the samples
After the plate plan is completed and saved, it is displayed in the "Planned Runs" panel of the main sequencing administrator page. The generated map of the run contains the volumes of template, primer, reaction mix and water to be added to each well. This map can be generated either for manual processing in the form of an Excel spreadsheet or as a CSV file for robotic processing. The two templates differ mainly in the volumes used.
To avoid reprogramming robotic systems for every run, a time consuming task which is prone to errors, all the modern liquid handling systems have the ability to load CSV files. As stated previously, PIMS can output the list of volumes of primers, water, buffer and templates in this format. Each of the three different liquid handling robots used within the University of Leeds sequencing facility requires a different output format, reflecting the different layouts of the decks of the robots. The sequencing extension can automatically generate 3 different types of CSV files for different robots and sequencing types. The most frequently used CSV file has fixed volumes for templates and primers, which are the same for all wells. This allows for faster sample processing using a time-optimised program. However, in some cases it is important to be able to vary the volumes of primer and template for each well. PIMS can prepare a differently formatted CVS file for such cases. The operator can choose whichever option is the most appropriate for the samples.
As the qualities of the DNA or the primers provided by the customer can vary, the required volumes might need to be changed to optimise the sequencing reaction. To allow the sequencing operator to address this problem, the variable volumes CSV files as well as the spreadsheet for manual processing can be amended. The modified files can then be uploaded into PIMS to provide a record of the alterations.
The system also prepares the task file for the sequencer, designated the "instrument set-up sheet". This sheet contains concatenated sample and primer names for each well, a unique run number and a unique reference number for each sample. The sample reference number is searchable and represents a unique reference to a condition in a particular well. Once the sequencing set-up sheet is uploaded, the PIMS considers the run to be at the "Awaiting Results" stage.
Completing the run
Once a prepared plate has been loaded into and processed by the sequencer, the sequencing results need to be uploaded into the PIMS system. The run is considered to be at "awaiting results" stage before this is done. The output of the sequencer consists of a set of files for each sample. It is possible to upload files for the same run one by one but for convenience the files are packed into a single ZIP archive which can then be uploaded into PIMS. The system then automatically links each result file from the archive with the sample information already recorded in the system. A "traffic lights" system of green, yellow and red colours respectively is used to indicate orders in which respectively all, some or no samples have been uploaded (Figure 4).
Upon uploading of the archive, the system rates the results either as OK, Failed or In_Process. OK means that the sequencing has been successful and the length of the sequence obtained is equal to or greater than that requested. Failed means that the sequencing has not been successful, or that the readable sequence length is shorter than that requested. In_Process marks the samples for which results have not been uploaded yet. The automatically assigned status can be manually altered by the facility operator. The result files marked Failed are not shown to the user, but can be preserved for future reference by the sequencing operator.
Apart from being able to download individual chromatogram and text files with the sequence for each sample (by clicking on seq, ab1 and scf file links), it is also possible to download all results at once. For example, option "fasta" exports all the sequences from the order as a FASTA formatted file. Option "trimmed", does the same, except it chops unresolved parts of the sequence from the ends, while option "archive", exports all the results that have been uploaded, including chromatogram files.
It is possible to annotate each sample with any number of notes, each of which bears the date and time when it was created and its author. These notes can either be visible to the sequencing operator only (private), or visible to everyone (public). Failed samples can either be tagged for re-injection or marked for re-processing if the cause of failure is unclear to the operator.
Once the sequencing operator has uploaded and scored all the results and added notes for the users, the plate can be completed. When the plate is completed the users are notified by email that their sequencing order is completed. The email contains a link to the web page where the results can be collected.
Data retrieval
Upon receipt of an email, informing the users of the completion of the sequencing order, they can retrieve their results from the system (Figure 5.). PIMS ensures data safety by restricting the users to their own results.
For each sample the user is provided with sequencing results statistics which allow a judgement on the quality of the sequencing to be made. Samples which sequencing failed can be submitted to re-processing at the click of a button, there is no need to manually re-enter the sample information into the system.