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===Interior Blast=== I also performed a blast on the templates in combolock (padlocks, positive control templates, and full sequencing primers) against the individual sequences, using the templates as the db. In the table below are the matches that were not expected (off-target matches). For this one I did not do any thresholding since I want to see all possible off-target bindings, although blast seems to have a threshold of at least 7 bases. Highlighted in the table below are the most concerning sequence matches (it's all the same similarity). That is the LinkerR region matches to somewhere on Primer2. It is a 9bp match, and while it is not very likely, is certainly possible to cause problems. blastn -db combolockseq.fa -query primerseqs.fa -task blastn-short -outfmt 7 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | width="127" height="30" | query id | width="98" | subject id | width="65" | % identity | width="64" | alignment length | width="70" | mismatches | width="65" | gap opens | width="65" | q. start | width="65" | q. end | width="65" | s. start | width="65" | s. end | width="65" | evalue | width="65" | bit score | width="89" | Notes |- style="background-color:#FFFF00;font-size:12pt" valign="bottom" | height="15" | primer2 | Padlock0001-68 | align="center" | 100 | align="center" | 9 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 13 | align="center" | 84 | align="center" | 76 | align="center" | 0.014 | align="center" | 18.3 | LinkerR* |- style="background-color:#FFFF00;font-size:12pt" valign="bottom" | height="15" | primer2 | AmpF6.4 | align="center" | 100 | align="center" | 9 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 13 | align="center" | 54 | align="center" | 46 | align="center" | 0.014 | align="center" | 18.3 | LinkerR* |- style="font-size:12pt" valign="bottom" | height="15" | primer6 | Amp3 | align="center" | 100 | align="center" | 8 | align="center" | 0 | align="center" | 0 | align="center" | 4 | align="center" | 11 | align="center" | 44 | align="center" | 51 | align="center" | 0.057 | align="center" | 16.4 | L3BC* |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | IlluminaP5 | Amp3 | align="center" | 100 | align="center" | 7 | align="center" | 0 | align="center" | 0 | align="center" | 4 | align="center" | 10 | align="center" | 61 | align="center" | 55 | align="center" | 0.26 | align="center" | 14.4 | Primer6* |- style="font-size:12pt" valign="bottom" | height="15" | IlluminaP5 | Amp1 | align="center" | 100 | align="center" | 7 | align="center" | 0 | align="center" | 0 | align="center" | 4 | align="center" | 10 | align="center" | 61 | align="center" | 55 | align="center" | 0.26 | align="center" | 14.4 | Primer6* |- style="background-color:#FFFF00;font-size:12pt" valign="bottom" | height="15" | LinkerR | Amp3 | align="center" | 100 | align="center" | 9 | align="center" | 0 | align="center" | 0 | align="center" | 15 | align="center" | 23 | align="center" | 13 | align="center" | 5 | align="center" | 0.023 | align="center" | 18.3 | Primer2* |- style="background-color:#FFFF00;font-size:12pt" valign="bottom" | height="15" | LinkerR | Amp1 | align="center" | 100 | align="center" | 9 | align="center" | 0 | align="center" | 0 | align="center" | 15 | align="center" | 23 | align="center" | 13 | align="center" | 5 | align="center" | 0.023 | align="center" | 18.3 | Primer2* |- style="font-size:12pt" valign="bottom" | height="15" | Latch3Barcode | Amp3 | align="center" | 100 | align="center" | 7 | align="center" | 0 | align="center" | 0 | align="center" | 2 | align="center" | 8 | align="center" | 71 | align="center" | 65 | align="center" | 0.057 | align="center" | 14.4 | Primer6* |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Latch3Barcode | Amp1 | align="center" | 100 | align="center" | 7 | align="center" | 0 | align="center" | 0 | align="center" | 2 | align="center" | 8 | align="center" | 71 | align="center" | 65 | align="center" | 0.057 | align="center" | 14.4 | Primer6* |}
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