Note: We assume quality control of the data has already been performed before using PrimerPipeline, and we recommend performing a de novo assembly with your data with, for example, using SOAPdenovo2.

• Step 1: Sequence Trimming and Finding Microsatellites

  This step incorporates sequence trimming and microsatellite mining (MISA).

  Sequence trimming removes ambiguous sequences and creates a minimum and maximum sequence length that will be used in microsatellite mining. Check that this minimum and maximum is suitable for your sequence data in the Settings tab.

  MISA will find microsatellites within your sequences from mononucleotides to hexanucleotide repeats ((A)30 to (TCACAC)6). In the Settings tab you can set the minimum repeat motif of microsatellites for each type of microsat.

  Automatically creates input file for Primer3.

• Step 2: Primer3

  PrimerPipeline incorporates Primer3 (Untergasser et al. 2012) which designs primers around your newly found microsatellites. You have the same functionality as using Primer3 directly, all settings can be adjusted to suit the user.

• Step 3: Creating the Results File

  When finished you can open a Results window, where the results are displayed in a user friendly way, allowing you to pick the primers you chose to optimise in an easy, well informed way. The forward and reverse complement primers, and the microsatellite are colour coded for easy visualisation of what you would be amplifying, and allows you to check your flanking regions. Your results file will be created in the same folder as your original input file.

For full details on how to use PrimerPipeline, read the user manual. This is available as part of the download, or can be read separately here.

This will vary based on your computer and on your data, how many microsatellites are contained within it, and the settings that you use. However, the Status column keeps you informed on PrimerPipeline's progress within each step, and presents a percentage complete. There are also Sequences read and SSRs found columns, which update during Step 1.