Link to this post | posted 02 Mar, 2024 22:24

ClaireRinehart

Debbie, I have additionally found Luxx gene 21 and 22 starts have been changed to bring this genome into "conformity" with the other EE genomes. Looking at the data I again disagree with these changes. Please educate me on the rationale. Thanks, Claire Rinehart Luxx Gene 21 (reverse gene) Starterator calls start 15583, which has a very poor Z- and Final Score but captures all of the coding capacity. Start 15577 is just six bases shorter and has a viable Z- and Final score, while capturing most all of the coding capacity. The best scoring start is at 15520 with excellent Z- and Final Scores but looses 63 bases of coding capacity found in the tail region of the typical plot, but into the peak region of the atypical plot. My choice is start 15577. Starterator Info for manual annotations of cluster EE: •Start number 3 was manually annotated 1 time for cluster EE. •Start number 5 was manually annotated 9 times for cluster EE. •Start number 6 was manually annotated 78 times for cluster EE. •Start number 7 was manually annotated 2 times for cluster EE. •Start number 10 was manually annotated 2 times for cluster EE. •Start number 12 was manually annotated 5 times for cluster EE. •Start number 14 was manually annotated 1 time for cluster EE. •Start number 15 was manually annotated 2 times for cluster EE. Gene: Luxx_21 Start: 15583, Stop: 15083, Start Num: 6 Candidate Starts for Luxx_21: (2, 15784), (Start: 5 @15595 has 9 MA's), (Start: 6 @15583 has 78 MA's), (Start: 7 @15577 has 2 MA's), (9, 15559), (Start: 10 @15553 has 2 MA's), (Start: 12 @15520 has 5 MA's), (16, 15475), (17, 15439), (18, 15424), (19, 15397), (20, 15385), (22, 15343), (23, 15319), (25, 15289), (28, 15259), (29, 15250), (30, 15229), (31, 15226), (33, 15202), (38, 15118 ), Ribosomal binding scores: Direction  Start   Stop  Length  Gap  Spacer  Z-score  Final Score  Codon Reverse    15784  15083   702   -122    14        2.391    -4.856         ATG Reverse    15595  15083   513      67      7         0.6         -8.437         GTG Reverse    15583  15083   501      79    12         0.503    -7.945         ATG Reverse    15577  15083   495      85    10         1.201    -6.398         ATG Reverse    15559  15083   477    103    10         0.549    -7.710         ATG Reverse    15553  15083   471    109    10         1.201    -6.398         ATG Referse    15520  15083   438    142    13         2.987    -3.155         ATG Luxx Gene 22 (reverse gene) (reverse gene) Starterator calls the start at 15893. As you can see below, start 15893 has one of the poorest Z- and Final Scores. A better choice would be 15923 or 15818. In looking at the coding capacity below, Start 15818 would give up a large portion of coding capacity. However, start 15923 would even capture the atypical coding capacity and is my start of choice. Starterator Gene: Luxx_22 Start: 15893, Stop: 15663, Start Num: 5 Candidate Starts for Luxx_22: (3, 15935), (4, 15923), (Start: 5 @15893 has 100 MA's), (6, 15818 ), (7, 15809), (8, 15791), (10, 15740), (11, 15728 ), Ribosomal binding scores: Direction  Start   Stop  Length  Gap  Spacer  Z-Score  Final Score Reverse    15935  15663    273    468    15     1.495     -6.714 Reverse    15923  15663    261    480    15     2.237     -5.221 Reverse    15893  15663    231    510    15     0.936     -7.839 Reverse    15818  15663    156    585      9     2.137      -4.595 Reverse    15809  15663    147    598    18     2.137     -6.122 Edited 03 Mar, 2024 10:55

Link to this post | posted 02 Mar, 2024 21:55

ClaireRinehart

Debbie, I was using the GenBank submission of Luxx (cluster EE) to evaluate the annotations of our student's practice genomes that are based on Luxx. I found that gene 18 had a -34 gap start called instead of the -4 that we originally called. Start  Stop  Length  Gap  Spacer  Z-score  Final Score  Codon  Forward 14090  14578    489 -259       6    0.875       -8.104         GTG   Forward 14315  14578    264  -34      16    1.587       -6.724         GTG   Forward 14327  14578    252  -22      10    1.637       -5.522         GTG   Forward 14345  14578    234   -4      16    2.066       -5.760         GTG   Forward 14426  14578    153   77       6    1.917       -6.007         ATG   Forward I read the Cluster EE forum notes and see that Luxx was modified to bring the group into "conformity". I would like to learn what would justify gene 18 start being called at 14315 other than the fact that the other 84 genomes in Starterator use that site? Thanks, I am still learning. Claire Edited 02 Mar, 2024 22:42

Link to this post | posted 22 Mar, 2022 14:31
ClaireRinehart	The NCBI BLAST is now operating and the backlog has been significantly reduced. Thanks again. Claire

Link to this post | posted 22 Mar, 2022 03:46
ClaireRinehart	Thanks Steve for point this out. You are right. It has been a week for some submissions. We are working on getting the backlog resolved. Claire

Link to this post | posted 02 Jul, 2021 16:52
ClaireRinehart	PECAAN has been modified to output the tRNA report so that it now passes the QC workflow.

Link to this post | posted 01 Jun, 2021 21:35

ClaireRinehart

PECAAN Annotation Tutorial Videos * Finding closest relatives https://youtu.be/5jqoHZwacAM * Compare genome to closest relatives https://youtu.be/6dh9yiWR2yw * How the locations of genes are predicted https://youtu.be/51YurlcyJKk * How to add and delete genes https://youtu.be/aNmH541DGMA * SEA PHAGES annotation guide https://youtu.be/4MYjl0T5cKY * Starterator, PhagesDB & NCBI BLAST https://youtu.be/85JwOLoBwFU * Gaps & Ribosomal Binding Sites https://youtu.be/dj-YcygwP3s * GM coding capacity, LORF & start sites https://youtu.be/L_AIQ1rAUxg * tRNA and tmRNA https://youtu.be/n07izcvyUGE * Assigning a function https://youtu.be/VV97ZP7ZpG0 PECAAN Admin Tutorial Videos * Why PECAAN? https://youtu.be/KlVepaPHA3g * How to put a Phage Genome into PECAAN https://youtu.be/Vxf9Bs1QysY * How to put Users into PECAAN https://youtu.be/3l1pMuMEXgg * How to update Official Function List https://youtu.be/wiKv9A0cX_c Edited 02 Jun, 2021 10:55

Link to this post | posted 11 Jul, 2020 23:59

ClaireRinehart

Well, here we are again. This time the Cluster A1 phage is STLscum and it has all of the features described above. I notice that there are now others that fit the criteria above that have called this superinfection immunity protein or superinfection exclusion protein including Swag_38, LastResort_38, Jabith_72, and Niza_72. All of these have 2-3 transmembrane domains and are good matches to pfam 14373. Would you reconsider naming this group of proteins "superinfection exclusion protein" after JSwag_38. This seems more appropriate since this protein contributes to the exclusion of super-infecting genomes to the periplasmic space? Thanks, Claire

Link to this post | posted 27 Jun, 2020 13:32

ClaireRinehart

Only 8 of the 109 have the -4 start site at Starterator location 12. As you can see from the Starterator map, all of these belong to the family of longest ORFs. All of the closest nucleotide BLAST relatives to OfUltron and Seabastion (Llama, Modragons and Ochi17) have this -4 start. I am going to call the -4 start because the data is consistent for these longer ORFs and may be a new evolving pham.

Link to this post | posted 26 Jun, 2020 21:54

ClaireRinehart

Welkin, I am struggling with the evidence for OfUltron and Sebastian gene 103. The -4 gap start is at 54324 and has a Z-score of 3 and Final score of -2.6 which looks like very compelling evidence for this start site. When I look at Starterator results I find that all 109 Cluster F1 hits call the start at 54471 which has a Z-score of 2.255 and a Final score of -4.458. When I look at the secondary structure potential for the RBS at start 54471 I find that 5 of the seven bases are in a very strong stem of a local stem-loop with a final Free Energy of -1200, which is very high (6 of the 7 bp in the stem are G-C bp). Wow, this makes the -4 gap look even better. However, if translation starts at the -4 start site there is no coding capacity for about 70 bases and then there is atypical coding capacity for about 50 bases before the start at 54471. I searched this non-coding capacity range and found 4 rare codons in this region. My initial instinct is to go with start 54324 with the -4 gap with the hope that some of the ribosomes would be able to navigate the rare codon domain, even though that may be at a slower rate. Is there Mass Spec data for Cluster F1 phages or other evidence (besides herd instinct) that has pointed everyone else to call the start at 54471?

Link to this post | posted 13 May, 2020 23:08

ClaireRinehart

Since PECAAN's BLASTS, HHPred and other queries are run locally in our High Performance Computer Center to avoid overloading NCBI and other services we sometimes get out of sync with them. We check these databases regularly to see when we need to update, but sometimes we get out of sync and simply need to download the latest database. So, if you see this kind of problem again, just drop us a line and we will initiate an additional download from these databases. HPCC administrator has informed me that we should be back in sync. Just a note concerning a global re-run on your whole phage from the Admin - Phages window that Chris mentioned. If you re-BLAST the whole phage genome you will often loose the evidence checkmarks. Therefore, do so with the assumption that the checkmarks will be reset to zero. Thanks, Claire Edited 13 May, 2020 23:10