SEA-PHAGES | All posts created by mdgainey

Link to this post \| posted 12 Oct, 2018 22:22
mdgainey	Hi I thought I would just add a bit to this thread since I had some issues with my preps last year. For our initial digest analysis this year I think I am just going to omit the DNase and RNase treatment altogether, and instead of precipitating the DNA I am going to do the 4 degree concentration spin that the guide suggests, and then prep/digest. I had good luck with this, this summer with some of my Microphages that I had issues with last semester and got nice clean yields. For the phages selected for sequencing, I will use the protocol as Vic has suggested. Just thought I would throw this out there as another option to try.

Link to this post | posted 12 Oct, 2018 22:22

Hi I thought I would just add a bit to this thread since I had some issues with my preps last year. For our initial digest analysis this year I think I am just going to omit the DNase and RNase treatment altogether, and instead of precipitating the DNA I am going to do the 4 degree concentration spin that the guide suggests, and then prep/digest. I had good luck with this, this summer with some of my Microphages that I had issues with last semester and got nice clean yields. For the phages selected for sequencing, I will use the protocol as Vic has suggested. Just thought I would throw this out there as another option to try.

Posted in: Phage Discovery/Isolation → DNA Isolation from M. foliorum - any updates?

Link to this post \| posted 18 Jul, 2018 19:08
mdgainey	Thus far it appears that none of the EA phages are reported to contain tRNAs. We recently finished annotating an EA2 bacteriophage Andromedas, that had a potential tRNA that was not called by Aragorn, but was called by tRNA scanSE but bc it had an infernal score of 38 and we decided to not include it in our annotation after looking at the structure of the anticodon loop. I am currently checking an EA5 phage, Neferthena, and again we found a potential tRNA that was not called by Aragorn, but was called by tRNAscanSE, this time the infernal score is 52. I have attached screen shots of the two tRNAs from PECAAN, and would like a second opinion on whether or not to keep the tRNA from Neferthena, the anti-codon loop does not look canonical to me, but I don't have much experience looking at these structures. Any advice is appreciated. Thank you, Maria 366Kb

Link to this post | posted 18 Jul, 2018 19:08

mdgainey

Thus far it appears that none of the EA phages are reported to contain tRNAs. We recently finished annotating an EA2 bacteriophage Andromedas, that had a potential tRNA that was not called by Aragorn, but was called by tRNA scanSE but bc it had an infernal score of 38 and we decided to not include it in our annotation after looking at the structure of the anticodon loop.

I am currently checking an EA5 phage, Neferthena, and again we found a potential tRNA that was not called by Aragorn, but was called by tRNAscanSE, this time the infernal score is 52.

I have attached screen shots of the two tRNAs from PECAAN, and would like a second opinion on whether or not to keep the tRNA from Neferthena, the anti-codon loop does not look canonical to me, but I don't have much experience looking at these structures.

Any advice is appreciated.
Thank you,
Maria

Posted in: tRNAs → tRNA in EA2 and EA5 phages called by tRNAscan SE but not aragorn

Link to this post \| posted 17 Jul, 2018 20:42
mdgainey	Thanks Welkin, Maria

Posted in: Frameshifts and Introns → New Frameshift in K5 bacteriophage RtcB-ligase ?

Link to this post \| posted 17 Jul, 2018 16:32
mdgainey	Thanks Welkin, Maria

Posted in: Functional Annotation → Cluster K5 terminases

Link to this post \| posted 17 Jul, 2018 15:50
mdgainey	I have been annotating a cluster K5 genome (Rando14). According to the functional assignments document, if we can only find 1 terminase, then we should simply label it terminase. However, a lot of the K's have called gene5 pham 40672(shown in attached powerpoint) a small terminase, but the HHPRED does not support this (attached). I took a look at old forum posts from some faculty working on calling cluster J terminases and found a post from Jacqueline Washington that suggested looking at this paper where they found a small terminase subunit that did not have an HTH binding motif, but did have a coiled coil motif " Archives of Virology 2012 Nov. 157 (11) in which they explained that most terminase small subunits have HTH DNA binding motifs but some don't. They used the program COILS2 (http://www.ch.embnet.org/software/COILS_form.html) to identify the small terminase as it reveals the presence of a coiled coil motif (CCM) which is need for protein dimerization." I ran gene 5 through this and it does indeed seem to have a nice CCM motif (output attached). Is this enough evidence to call this a small terminase subunit, or am I just following the leader here? Thank you, Maria 458Kb

Link to this post | posted 17 Jul, 2018 15:50

mdgainey

I have been annotating a cluster K5 genome (Rando14). According to the functional assignments document, if we can only find 1 terminase, then we should simply label it terminase. However, a lot of the K's have called gene5 pham 40672(shown in attached powerpoint) a small terminase, but the HHPRED does not support this (attached). I took a look at old forum posts from some faculty working on calling cluster J terminases and found a post from Jacqueline Washington that suggested looking at this paper where they found a small terminase subunit that did not have an HTH binding motif, but did have a coiled coil motif " Archives of Virology 2012 Nov. 157 (11) in which they explained that most terminase small subunits have HTH DNA binding motifs but some don't. They used the program COILS2 (http://www.ch.embnet.org/software/COILS_form.html) to identify the small terminase as it reveals the presence of a coiled coil motif (CCM) which is need for protein dimerization." I ran gene 5 through this and it does indeed seem to have a nice CCM motif (output attached). Is this enough evidence to call this a small terminase subunit, or am I just following the leader here?
Thank you,
Maria

Posted in: Functional Annotation → Cluster K5 terminases

Link to this post \| posted 13 Jul, 2018 18:22
mdgainey	Hi I have been annotating Rando14, a K5 bacteriophage and when we go to the area where many of the K5's have an RtcB ligase, there were some weird reverse genes that we needed to delete, and then we found 2 forward ORFs that we think should fill this area nicely with good CP, the first in the +2, and the second in the +1 reading frame that blast up to the N and C terminal portions of the the RtcB ligase respectively. I have attached a power point showing the potential slippery GGGG sequence, where I think the frame shift might occur, and the protein products. Do you think this could possibly be a sequencing error, or perhaps a real frameshift? I know we don't have wet bench data to support frameshifts, other than in the tail assembly chaperones, so I would probably not annotate this as such, but since this one has a predicted function I thought I would post about it to get some advice on how to handle this one. Thank you, Maria 324Kb

Link to this post | posted 13 Jul, 2018 18:22

mdgainey

Hi I have been annotating Rando14, a K5 bacteriophage and when we go to the area where many of the K5's have an RtcB ligase, there were some weird reverse genes that we needed to delete, and then we found 2 forward ORFs that we think should fill this area nicely with good CP, the first in the +2, and the second in the +1 reading frame that blast up to the N and C terminal portions of the the RtcB ligase respectively. I have attached a power point showing the potential slippery GGGG sequence, where I think the frame shift might occur, and the protein products.

Do you think this could possibly be a sequencing error, or perhaps a real frameshift?
I know we don't have wet bench data to support frameshifts, other than in the tail assembly chaperones, so I would probably not annotate this as such, but since this one has a predicted function I thought I would post about it to get some advice on how to handle this one.
Thank you,
Maria

Posted in: Frameshifts and Introns → New Frameshift in K5 bacteriophage RtcB-ligase ?

Link to this post \| posted 11 Dec, 2017 14:27
mdgainey	Dan Russell mdgainey Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem. Maria Hey Maria, I don't really have any good tips to reduce the host DNA, but my sense is I'd try sequencing it and see what comes out. If the host DNA really is overwhelming, then it's back to the drawing board, but I think there's a decent chance the phage sequence would stand out from the bacterial chunks, and then you'd be done. So I'd just try prepping and sequencing without DNAse and see what comes out, then only worry about reducing the host DNA if that first attempt didn't work. –Dan Thanks Dan, I will give it a go next semester. Maria

Link to this post | posted 11 Dec, 2017 14:27

mdgainey

Dan Russell
mdgainey
Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

Hey Maria,

I don't really have any good tips to reduce the host DNA, but my sense is I'd try sequencing it and see what comes out. If the host DNA really is overwhelming, then it's back to the drawing board, but I think there's a decent chance the phage sequence would stand out from the bacterial chunks, and then you'd be done. So I'd just try prepping and sequencing without DNAse and see what comes out, then only worry about reducing the host DNA if that first attempt didn't work.

–Dan

Thanks Dan, I will give it a go next semester.
Maria

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 10 Dec, 2017 22:20
mdgainey	Dan Russell Nicholas Edgington Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results. Hey Nick, We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows? Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help. –Dan Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem. Maria

Link to this post | posted 10 Dec, 2017 22:20

mdgainey

Dan Russell
Nicholas Edgington
Dan: With such great sequence coverage that has been seen from past years, would the contaminating host DNA really cause much of a problem with assembly? Maybe one could just add a bit of RNAse directly to the HTL's? It would be interesting to know if you have ever tried phage genomic sequencing with and without the addition of the DNAse/RNAse cocktail, and compared the assembly results.

Hey Nick,

We haven't tried that in any real experimental way. We do sometimes get samples with host DNA contamination, and in most cases it's relatively low coverage compared to the phage and thus isn't a problem. On the other hand, we have seen a few samples where the host coverage was high enough to drop the phage coverage to an unusable/undetectable level. So I guess: who knows?

Also, has anyone tried side-by-side old protocol versus new protocol to check for yields from each? That would perhaps shed some light. Having a high titer is certainly the most important thing to getting good yield, so bumping up the titer wherever possible is probably the best way to help.

–Dan

Thanks for posting this Nick. Dan, I have one podo with an unstable capsid and would like to try to seq/assemble with just an RNase treatment. Dan do you have any basic tips to reduce host DNA contamination in my prep to hopefully achieve a successful assembly without DNase treatment, sounds like it might be fine but if I could stack the deck a bit that would be great. This one grows really well so high titer should not be a problem.
Maria

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 10 Dec, 2017 22:10
mdgainey	Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

Link to this post | posted 10 Dec, 2017 22:10

mdgainey

Hi all, I posted a couple of weeks ago in the M. foliorum RE digests group, but just saw this thread. I used the Wizard kit for M. smeg phages in the past using the new protocol (however I used some nucleases I had already purchased not the recommended mix) and always had trouble with the nucleases coming through the prep. So basically after nuclease treatment but before the DNA prep I always treat my lysates with sds/protK/edta-55deg for 1 hr (before class and then freeze) and then had the students follow the protocol as normal, and it always worked great. This year I did the same thing but am having issues with about half the M. foliorum and couple of G. Terrae phages we have found after placing them at 37 degrees for digest. More disturbing was that I had the exact same issue when I had the students do nuclease free preps, even with sds/protK/edta treatments before the kit…so I am not sure what is going on. Could it be these phages are bringing a nuclease with them that is even harder to inactivate than the traditional DNases we normally use? I don't think this is a reagent issue as about half of the phages DNA that I put through the preps this year came out looking pristine, and this same procedure has worked like clockwork for me in the past.

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

Link to this post \| posted 17 Oct, 2017 14:18
mdgainey	Thanks Vic, I did not include the nucleases in this round of preps bc I have already run into an unstable capsid (DNA fully degraded before RE digest). After precipitation and prep, the DNA looks fine for the other phages that I have not added nucleases to, but as you said after adding buffer and placing at 37 degrees I see degradation. It seems that the phages that are cut by the traditional enzymes are the ones that I am seeing the degradation with. I will know next week how the rest of the class's preps look with the nuclease inactivation step added. I have never had good luck getting rid of the nucleases with the resin from the Wizard kit, and normally do the nuclease inactivation step with every prep that we do that nucleases have been added. I will have the students do an NspI digest and post that to phages db. Maria

Link to this post | posted 17 Oct, 2017 14:18

mdgainey

Thanks Vic,
I did not include the nucleases in this round of preps bc I have already run into an unstable capsid (DNA fully degraded before RE digest). After precipitation and prep, the DNA looks fine for the other phages that I have not added nucleases to, but as you said after adding buffer and placing at 37 degrees I see degradation. It seems that the phages that are cut by the traditional enzymes are the ones that I am seeing the degradation with. I will know next week how the rest of the class's preps look with the nuclease inactivation step added. I have never had good luck getting rid of the nucleases with the resin from the Wizard kit, and normally do the nuclease inactivation step with every prep that we do that nucleases have been added. I will have the students do an NspI digest and post that to phages db.

Maria

Posted in: Microbacterium → Restriction Enzyme Digests

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