Trouble that could not be designed with the defined parameters

[HFyange]

Hi, @aakechin

1. NGS-PrimerPlex version is the latest v1.3.4.
2. Warning: 16 regions primers could not be designed with the defined parameters. Here shows the regions:
   {
   PTENexon8_14
   TTGTTGACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAAT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_15
   TGTTGACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATA
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_16
   GTTGACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATAT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_17
   TTGACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATC
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_18
   TGACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_19
   GACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTA
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_20
   ACTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAG
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_21
   CTTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_22
   TTTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTA
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_23
   TTTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTAC
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_24
   TTTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_25
   TTGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_26
   TGCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTA
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_27
   GCAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTAC
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_28
   CAAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTACT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   PTENexon8_29
   AAATGTTTAACATAGGTGACAGATTTTCTTTTTTAAAAAAATAAAACATCATTAATTAAATATGTCATTTCATTTCTTTTTCTTTTCTTTTTTTTTTTTTTTAGGACAAAATGTTTCACTTTTGGGTAAATACATTCTTCATACCAGGACCAGAGGAAACCTCAGAAAAAGTAGAAAATGGAAGTCTATGTGATCAAGAAATCGATAGCATTTGCAGTATAGAGCGTGCAGATAATGACAAGGAATATCTAGTACTTACTT
   considered 0 pairs, ok 0;
   Left: considered 2236, not in any ok left region 2236, ok 0
   Right: considered 2236, not in any ok right region 2236, ok 0
   You can try to increase -primernum1 for this region
   }
3. It shows 16 regions. But I see that all these regions refer to PTENexon8 and have almost same sequence that should be as one region rather than 16 regions. Is this a problem?
4. {Left: considered 2236, not in any ok left region 2236, ok 0}. what is this meaning? why always 2236 number?

[aakechin]

@HFyange it shows all positions that are not covered because it tries to design primers pairs for each position that user defined. After that, it chooses best primer pairs. So, in your case it shows that positions from 14 to 29 were not covered by the designed primers. To design primers for the left positions, use -draft parameter with draft-file that was created at the current step, and change parameters of primer design.