Genomics Application Notes
Results and Discussion
Setting up Library Construction Automated library construction using 96 samples was set up by making a few selections in the Biomek FX P user interface (Figures 1 and 2). The user selects either the TruSeq Stranded mRNA method or the TruSeq RNA v2 method; this application note is focused on the TruSeq Stranded mRNA method. The automation method is flexible, allowing the user to choose which steps to perform. Users also have the option to use custom plates for the adaptor labware, and to choose which adaptors to use with which samples, by selecting their own transfer files. Setting up the PCR reaction to enrich for fragments with the proper adaptors is also optional (Figure 1). Once all of the method parameters have been entered, the Biomek FX P software provides a reagent calculator that tells the users the volume of each reagent needed, as well as how to prepare the reagents, when necessary (Figure 2).
Figure 1. Intuitive user interface enables quick and flexible setup of runtime parameters
Figure 2. Required reagent volumes are automatically calculated and displayed to help users avoid errors.
Sample Layout The RNA samples to be sequenced were placed in a 96 well plate configured as shown in Table 2 to assess cross contamination between wells. The library sequences from various species can be differentiated after sequencing using the individual index sequences added during the adaptor ligation step of library preparation. If cross contamination existed, we should be able to detect its presence by mapping the reads of a particular library against the reference genomes of libraries prepared in adjacent wells. Except for the tomato libraries in columns 11 and 12 of the library construction plate, all libraries contained 1ug of total RNA. Tomato libraries in columns 11 and 12 began library construction with 3ug of total RNA.
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