The run has met the Illumina specifications when the run metrics are within the thresholds and are colored green.
Yes. The MiSeq FGx Control Software offers an RUO run option alongside the Forensic Genomics run options. The RUO side of the software is fully compatible with all Illumina-supported RUO run types, and runs are set up as for standard MiSeq runs. For more details on set-up and workflow, see the MiSeq FGx Instrument Reference Guide.
Multiple samples can be pooled together because of the use of unique index (barcode) sequences added during library preparation. Indexes allow the software to separate out the data for each individual sample after sequencing.
Final pooled libraries can be stored at -25°C to -15°C for up to 3 months.
Preparing libraries takes approximately 9 hours. Sequencing and Real-Time Analysis (RTA) software tasks on the MiSeq FGx such as image processing, assigning base calls, and then designating a quality score for each base call takes approximately 30 hours. Aligning reads to provide sample information, along with optional steps such as generating reports, population statistics, sample comparison, phenotype estimations and bio-geographical ancestry estimations take approximately 1 hour.
To log in to MiSeq FGx Control Software, a connection must be established with the ForenSeq Universal Analysis Software server to check authentication. After authentication, an RUO run can be performed without connection to the server. There is a buffering mechanism with the MiSeq FGx Control Software to allow data transmission to resume upon a connection loss to ForenSeq Universal Analsyis Software during acquisition.
No. However, a sample sheet created using IEM is compatible with the RUO mode of the MiSeq FGx Control Software. For forensic runs, the samples are defined in the ForenSeq Universal Analysis Software before acquisition through a file upload or manual entry. For details on the information in the file, see the ForenSeq Universal Analsyis Software Guide.
An account can be set up on the ForenSeq Universal Analysis Software. See the ForenSeq Universal Analysis Software Guide for more details. This account is used on the sequencing instrument to log in to MiSeq FGx Control Software and start the runs created by the account holder on the ForenSeq Universal Analysis Software.
The post-run wash takes 30 minutes and a maintenance wash takes about 1 hour.
Yes, the MiSeq FGx needs must be washed between runs to remove any remaining reagents from the fluidics lines and make sure that they are clean before the next run is started. The MiSeq FGx offers a bleach post-run wash protocol that minimizes run-to-run contamination. See the MiSeq FGx Instrument Reference Guide for wash instructions.
Yes, the same metrics that are shown during data acquisition are available on the ForenSeq Universal Analysis Software after the run is complete.
Yes, in order for the results to be transferred from the MiSeq FGx to the ForenSeq Universal Analysis Software a connection must exist between the systems. In many cases that connection is established through the company network. However, in some labs that connection can be on a local network created with the provided router.
Data analysis is performed using the ForenSeq Universal Analysis Software.
Yes. Direct sample to sample comparison can be performed using the ForenSeq Universal Analysis software.