SEA-PHAGES | Tricky Start position decision: Need 2nd Opinion; Two overlapping Genes with Strong CP: MrMiyagi

Link to this post \| posted 05 May, 2020 19:23
fbaliraine	Having trouble deciding on current Genes 42 and 43 in Phage MrMiyagi. Attached, please find snapshots of RBS scores, Frames, Coding potential, and Phamerator maps. These two gens apparently have overlapping ORFs. No CP seen in GeneMark-smeg & TB, but strong coding potential for both in GeneMarkS. Start for gp 43 (45 in pharmerator) called by GL & in Cuke & Fowlmouth; staterator; SSC: 35734; Z= 1.676, F= -6.512; spacer distance 16; with 6 bp overlap; we were selecting the start at 35716, Z= 2.487, F= -5.050, 26 bp overlap with gene 42 (44 in phamerator); with a shorter spacer distance of 5, but realize this gene 43 ORF is continuous from 35449 bp (Z= 2.018, F= -5.536; the next start is with a better RBS at 35572 bp with Z= 2.673, F= -3.504). The short gene 43 (45 in phamerator) apparently currently has only 3 members in the pham 32826; compared to 44 which has 61 members (pharm 40615). Start for gene 42 (44 in phamerator) is inside CP plateau, hence not covering all CP, but it’s the 1st one; see CP figure attached. Since there is no break/stop codon in the ORF for gene 43(45 in phamerator) and there’s strong RBS scores for the 1st and 3rd starts which are overlapping gene 42 (44 in phamerator) is there no chance that this is the same gene? Should I delete 42 (44 in phamerator) and take the start at 35572 bp with Z= 2.673, F= -3.504, or should I just overlook this, keep gene 42 (44 in phamerator) and take the start called by GL at 35734; z= 1.676, F= -6.512 and in Cuke & Fowlmouth 6bp overlap, or the one I am considering with a better RBS score at 35716, Z= 2.487, F= -5.050, 26 bp overlap? Please advise. Edited 06 May, 2020 16:22 734Kb

Link to this post | posted 05 May, 2020 19:23

Having trouble deciding on current Genes 42 and 43 in Phage MrMiyagi. Attached, please find snapshots of RBS scores, Frames, Coding potential, and Phamerator maps. These two gens apparently have overlapping ORFs. No CP seen in GeneMark-smeg & TB, but strong coding potential for both in GeneMarkS. Start for gp 43 (45 in pharmerator) called by GL & in Cuke & Fowlmouth; staterator; SSC: 35734; Z= 1.676, F= -6.512; spacer distance 16; with 6 bp overlap; we were selecting the start at 35716, Z= 2.487, F= -5.050, 26 bp overlap with gene 42 (44 in phamerator); with a shorter spacer distance of 5, but realize this gene 43 ORF is continuous from 35449 bp (Z= 2.018, F= -5.536; the next start is with a better RBS at 35572 bp with Z= 2.673, F= -3.504). The short gene 43 (45 in phamerator) apparently currently has only 3 members in the pham 32826; compared to 44 which has 61 members (pharm 40615). Start for gene 42 (44 in phamerator) is inside CP plateau, hence not covering all CP, but it’s the 1st one; see CP figure attached.
Since there is no break/stop codon in the ORF for gene 43(45 in phamerator) and there’s strong RBS scores for the 1st and 3rd starts which are overlapping gene 42 (44 in phamerator) is there no chance that this is the same gene? Should I delete 42 (44 in phamerator) and take the start at 35572 bp with Z= 2.673, F= -3.504, or should I just overlook this, keep gene 42 (44 in phamerator) and take the start called by GL at 35734; z= 1.676, F= -6.512 and in Cuke & Fowlmouth 6bp overlap, or the one I am considering with a better RBS score at 35716, Z= 2.487, F= -5.050, 26 bp overlap? Please advise.

Edited 06 May, 2020 16:22

Link to this post \| posted 06 May, 2020 18:54
welkin	Hi Fred, Thanks for your detailed documents; it makes helping you much easier. First: You have two genes. The reasons you should not delete 42 is it has better coding potential than 43 and it is present in at least one other phage (Fowlmouth– as you pointed out). So: now that you are keeping 42; you have to use a start that does not capture all the CP of 43, as that would lead to a substantial overlap with 42 without a good reason (good reasons include high quality function alignments). The best start is the most conserved start— again, the one annotated in the other phage and selected by Glimmer; visible in Starterator. Both of those things absolutely outweigh the RBS score (both scores are pretty bad, honestly, so it is hard to consider it at all). I hope that helps. Best, Welkin Edited 06 May, 2020 23:05

Link to this post | posted 06 May, 2020 18:54

welkin

Hi Fred,
Thanks for your detailed documents; it makes helping you much easier.
First: You have two genes.
The reasons you should not delete 42 is it has better coding potential than 43 and it is present in at least one other phage (Fowlmouth– as you pointed out).

So: now that you are keeping 42; you have to use a start that does not capture all the CP of 43, as that would lead to a substantial overlap with 42 without a good reason (good reasons include high quality function alignments).

The best start is the most conserved start— again, the one annotated in the other phage and selected by Glimmer; visible in Starterator.
Both of those things absolutely outweigh the RBS score (both scores are pretty bad, honestly, so it is hard to consider it at all).
I hope that helps.
Best,
Welkin

Edited 06 May, 2020 23:05

Link to this post \| posted 06 May, 2020 21:59
fbaliraine	Thanks a lot Welkin! Fred

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Tricky Start position decision: Need 2nd Opinion; Two overlapping Genes with Strong CP: MrMiyagi