The Primer Walking Report

The Primer Walking report lists the selected primers in a tabular text format showing the sequence and other selection criterion. The report is generated using the Contig > Primer Walk command.

Double clicking on a row in the report directs the focus to the Alignment View and scrolls to the primer location.

The following columns are available in the Primer Walking report:

•      Show - A check in this column causes a display of the primer and its false priming sites to be displayed in the alignment window adjacent to the consensus. The locations are marked by red highlight for the top strand or green highlight for the bottom strand locations. The check also causes the primer to be included when the primer report is exported to file.

•      Score - A composite score combining weighted measures of desired location, Tm, assembly quality and mispriming, then scaled from 0 to 100 with higher being better.

•      Index - An ordinal number assigned to primers arbitrarily by location. Primers that share an index value are alternatives for the same location.

•      Primer sequence - The primer sequence is shown 5’-3’, prepared for synthesis requisition. Collapsing the field horizontally results in the sequence folding out its middle.

•      Contig (number) - The contig number that this primer extends or binds.

•      Strand - Bottom or top.

•      Start - The left end, 5' when top strand, 3' when bottom strand.

•      End - The right end, 3' when top strand, 5' when bottom strand.

•      Length - The length of the primer in nucleotides.

•      Tm - The melting temperature of the primer as computed by the nearest neighbor method.

•      GC Tm - The melting temperature of the primer as computed by the GC content method.

•      Delta G - The free energy of the 3'-end dimer (pentamer).

•      W/C - The purpose for which this primer was selected, W for walking, C for coverage.

•      Distance - The distance from the corresponding end of the contig. For top strand primers, it’s the distance from the 3’ end. For bottom strand primers, it’s the distance from the 5’ end.

•      Coverage - The number of sequence reads to which this primer could bond, considering only the sequence that is exposed after trimming.

•      False Sites - The number of locations that the primer could misprime based upon its 3’ uniqueness. All locations in all of the contigs are considered.

•      Locations - The list of locations of mispriming sites, showing contig, strand and position.

To copy an active Primer Report as tab-delimited text, choose Edit > Select All and then Edit > Copy.

To save an active Primer Report as a text file, select File > Save Primer Info. Choose one of the following options from the Save As Type (Win) or Format (Mac) pull down menus:

•      Tab-Separated - Saves all of the primers in the report as a .txt file.

•      Tab-Separated - only checked - Saves a .txt file of only those primers in the report that have a checkmark in the Show column.

•      FASTA Sequences - Saves all of the primers in the report as a .fas file.

•      FASTA Sequences - only checked - Saves a .fas file of only those primers in the report that have a checkmark in the Show column.