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.