SEA-PHAGES | All posts created by uOttawaPHAGE

Link to this post \| posted 10 Feb, 2022 05:28
uOttawaPHAGE	thanks Mitch. I also know little about RNA binding proteins, but given the HHPred results we will investigate if key residues are conserved. Hfq is a phage protein, so the idea that this protein and Hfq evolved from a common ancestor seems possible? I'm not sure if convergence vs. common descent is the simpler explanation. However, I haven't looked carefully at the sequence similarity (or lack of it).

Link to this post | posted 10 Feb, 2022 05:28

thanks Mitch. I also know little about RNA binding proteins, but given the HHPred results we will investigate if key residues are conserved. Hfq is a phage protein, so the idea that this protein and Hfq evolved from a common ancestor seems possible? I'm not sure if convergence vs. common descent is the simpler explanation. However, I haven't looked carefully at the sequence similarity (or lack of it).

Posted in: Annotation → New function? Hfq RNA binding protein

Link to this post \| posted 10 Feb, 2022 04:08
uOttawaPHAGE	Hi Debbie. We'll use RNA binding protein, but I'll also have the students gather more evidence to see if they can differentiate between the different RNA binding proteins. This is an annotation we will change in all the AZ phages that have this gene as we complete the AZ harmonization.

Posted in: Annotation → New function? Hfq RNA binding protein

Link to this post \| posted 04 Feb, 2022 21:02
uOttawaPHAGE	We may be purchasing a MINion nanopore sequencer and I was looking for details on what we'll need. I know Dan has done some Nanopore sequencing, but I assume some others have as well. Any suggestions would be great. Some questions: 1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information E7180S (24 rxns) 2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore: https://store.nanoporetech.com/ligation-sequencing-kit.html 3. And these barcodes for up to 24 samples: https://store.nanoporetech.com/native-barcoding-expansion-1-12.html https://store.nanoporetech.com/native-barcoding-expansion-13-24.html 4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume? 5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready…. 6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Link to this post | posted 04 Feb, 2022 21:02

uOttawaPHAGE

We may be purchasing a MINion nanopore sequencer and I was looking for details on what we'll need. I know Dan has done some Nanopore sequencing, but I assume some others have as well. Any suggestions would be great. Some questions:

1. Do you use the NEB ligation kit? NEBNext companion module for Oxford nanowire technologies ligation sequencing https://international.neb.com/products/e7180-nebnext-companion-module-for-oxford-nanopore-technologies-ligation-sequencing#Product%20Information
E7180S (24 rxns)

2. We were going to avoid the Q20 kits until they are more widely used. So I'm assuming we need this kit from oxford nanopore:

https://store.nanoporetech.com/ligation-sequencing-kit.html

3. And these barcodes for up to 24 samples:

https://store.nanoporetech.com/native-barcoding-expansion-1-12.html

https://store.nanoporetech.com/native-barcoding-expansion-13-24.html

4. Similar question as on my MiSeq post: has anyone tried using these kits at half volume?

5. Will they ship the first flow cell separately from the device? Dan mentioned flow cell aging is an issue, but we may have to spend the money we have before we have the libraries are ready….

6. Someone mentioned to me the flow cells can be reused, which seemed confusing given what Dan had mentioned about old flow cells. If they are cleaned and reused in a short time, will that work? This would be a great feature for using it in our course.

Posted in: Sequencing, Assembling, and Finishing Genomes → Nanopore sequencing questions

Link to this post \| posted 04 Feb, 2022 20:28
uOttawaPHAGE	We are annotating JohnDoe, a cluster AZ phage. Gene 53 in related phages has been annotated as NKF (by us and others), but we see high probability on HHpred for similarity to Hfq RNA binding proteins. The only two RNA binding protein options on the approved list are "RNA binding protein" and "Ro RNA binding protein". Can this new function be added? See attached doc. All other AZ phage with this gene will need to be corrected. thanks! Adam 233Kb

Link to this post | posted 04 Feb, 2022 20:28

uOttawaPHAGE

We are annotating JohnDoe, a cluster AZ phage. Gene 53 in related phages has been annotated as NKF (by us and others), but we see high probability on HHpred for similarity to Hfq RNA binding proteins. The only two RNA binding protein options on the approved list are "RNA binding protein" and "Ro RNA binding protein". Can this new function be added?

See attached doc.

All other AZ phage with this gene will need to be corrected.

thanks!
Adam

Posted in: Annotation → New function? Hfq RNA binding protein

Link to this post \| posted 04 Feb, 2022 19:19
uOttawaPHAGE	We are annotating a gene with a HTH DNA binding domain. However in the notes column of the approved list it refers to the function HTH DNA binding protein, even though only HTH DNA binding domain is on the approved function list. I'm pretty sure you consider these two things to be the same, but it was confusing to my students. Do you consider domain and protein different annotations?

Posted in: Annotation → helix-turn-helix binding domain or protein?

Link to this post \| posted 31 Jan, 2022 20:43
uOttawaPHAGE	thanks Dan. The library cost is clearly what adds up, which is why the 1/2 vol question. We will probably try that for a reaction or two. Sounds like you use the NEB beads?

Posted in: Sequencing, Assembling, and Finishing Genomes → MiSeq protocol and info

Link to this post \| posted 26 Jan, 2022 17:41
uOttawaPHAGE	We will be able to do a MiSeq run later this term, but we need to prepare the library and purchase the reagents. Ideally we'd like to do this the same way it is done at Pitt, so this is largely directed at Dan and Becky, but anyone who has done MiSeq sequencing, please feel free to comment too. 1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads? 2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY? 3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money. 4. Do you use the Illumina v2 reagent kit? 5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit? 6. I believe Pitt uses a Qbit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings? 7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….). 8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached. 498Kb

Link to this post | posted 26 Jan, 2022 17:41

uOttawaPHAGE

We will be able to do a MiSeq run later this term, but we need to prepare the library and purchase the reagents. Ideally we'd like to do this the same way it is done at Pitt, so this is largely directed at Dan and Becky, but anyone who has done MiSeq sequencing, please feel free to comment too.

1. Is this the library kit? NEB #E7805S Any opinions about using NEB SPRIselect vs. AMPure XP beads?

2. Do you use dual or single indexed adaptors/barcodes? If single indexed, do you purchase a 96 barcode kit (NEB E7335L)? Or are there 48 barcode kits? Or can this be DIY?

3. For library construction, can reactions be done in half volume? We tried this for a IonTorrent library and it worked fine. Obviously not the manufacturer's instructions, but it would save us quite a bit of money.

4. Do you use the Illumina v2 reagent kit?

5. Any other consumables needed? Is there a distinct wash kit or does it come with the reagent kit?

6. I believe Pitt uses a Qbit to assess the library. We have access to one, but I've not used it, Are there special reagents needed? Or specific settings?

7. Any details on the MiSeq settings? Are you running 2X150bp reads? I will have help from someone who has sequenced yeast genomes, so his settings may be different (if there are other settings….).

8. Someone sent me a DIY protocol for the AMPure beads. Any experience making them? We'll purchase some for this project, but long-term we would consider this. See attached.

Posted in: Sequencing, Assembling, and Finishing Genomes → MiSeq protocol and info

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