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Link to this post \| posted 31 Oct, 2024 16:58
DanRussell	Hi Tammy, This is a common question. I'm actually working on updating the PhagesDB database to include type of sequencer, but in the meantime here's a shorthand: If your phage was sequenced by Illumina at Pitt before February 2024, it was on an Illumina MiSeq. If it was March 2024 or after, it was on an Illumina NextSeq 1000. Similarly, if your phage was sequenced on the MiSeq, we recommend adding "Raw" to make your sentence "Raw reads were assembled…" because for phages run on the MiSeq, we haven't done any additional trimming or QC. This will change for the NextSeq 1000 genomes, so if your phage genome was sequenced on that, the reads were trimmed and filtered by: cutadapt 4.7 (using the option: –nextseq-trim 30) and skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50) Versions of other programs: Newbler 2.9 Consed 29 For genomic termini, it's too complicated to describe without using most of the 500 words of your MRA, so I usually tell people to just say "as previously described" and cite the chapter I wrote about precisely that. https://pubmed.ncbi.nlm.nih.gov/29134591/ Hope that helps! Oh, and don't forget the "h" in Pittsburgh! –Dan Edited 31 Oct, 2024 17:00

Link to this post | posted 31 Oct, 2024 16:58

Hi Tammy,

This is a common question. I'm actually working on updating the PhagesDB database to include type of sequencer, but in the meantime here's a shorthand:

If your phage was sequenced by Illumina at Pitt before February 2024, it was on an Illumina MiSeq. If it was March 2024 or after, it was on an Illumina NextSeq 1000.

Similarly, if your phage was sequenced on the MiSeq, we recommend adding "Raw" to make your sentence "Raw reads were assembled…" because for phages run on the MiSeq, we haven't done any additional trimming or QC. This will change for the NextSeq 1000 genomes, so if your phage genome was sequenced on that, the reads were trimmed and filtered by:

cutadapt 4.7 (using the option: –nextseq-trim 30)
and
skewer 0.2.2 (using the options: -q 20 -Q 30 -n -l 50)

Versions of other programs:
Newbler 2.9
Consed 29

For genomic termini, it's too complicated to describe without using most of the 500 words of your MRA, so I usually tell people to just say "as previously described" and cite the chapter I wrote about precisely that.
https://pubmed.ncbi.nlm.nih.gov/29134591/

Hope that helps!

Oh, and don't forget the "h" in Pittsburgh!

–Dan

Edited 31 Oct, 2024 17:00

Posted in: Sequencing, Assembling, and Finishing Genomes → MRA required information

Link to this post \| posted 26 Sep, 2024 14:29
DanRussell	Hi Stephanie, I don't think we ever finalized the sequence of this one, but we do have some Illumina reads which we assembled into WGS contigs. I'll add it to our Nanopore sequencing queue and run it the next time we do a MinION run so we can get the complete sequence. In the meantime, if you'd like to have the contigs we've assembled, I can email those to you. –Dan

Posted in: Arthrobacter → Genome Sequence Arthrobacter globiformis NRRL B-2979

Link to this post \| posted 04 Jun, 2024 17:04
DanRussell	And another one from Roger Hendrix, from this paper: Comparative examination of [phage] genomes indicates that the hallmark of phage evolution is horizontal exchange of sequences. This is accomplished, first, by rampant non-homologous recombination between different genomes and, second, by reassortment of the variant sequences so created through homologous recombination.

Posted in: General Message Board → Classificiation with ICTV guidelines

Link to this post \| posted 04 Jun, 2024 17:02
DanRussell	I think Graham would probably point out that phages don't really have "lineages" in the traditional sense, which is one of the reasons we don't systematically name them——or use ICTV classifications. Phage genetic material isn't only shared vertically (between different generations), but horizontally as well. Thus, trying to shoehorn phages into classification systems designed for other types of organisms doesn't work well. From a review article Graham wrote in 2008 (this paper): "One of the most striking features of bacteriophage genomes is their apparent mosaic structure; in essence, each genome can be considered as a unique combination of modules that are exchangeable among the population. The size of the modules, their rates of exchange, and the phage genomes carrying them all vary greatly, with phages of different virion morphology, size, and host-range all participants in an orgy of recombination [36]." Hence the advice above. Just explain some version of the above to the reviewer, and let GenBank/ICTV apply whatever designations they see fit!

Link to this post | posted 04 Jun, 2024 17:02

DanRussell

I think Graham would probably point out that phages don't really have "lineages" in the traditional sense, which is one of the reasons we don't systematically name them——or use ICTV classifications. Phage genetic material isn't only shared vertically (between different generations), but horizontally as well. Thus, trying to shoehorn phages into classification systems designed for other types of organisms doesn't work well.

From a review article Graham wrote in 2008 (this paper):

"One of the most striking features of bacteriophage genomes is their apparent mosaic structure; in essence, each genome can be considered as a unique combination of modules that are exchangeable among the population. The size of the modules, their rates of exchange, and the phage genomes carrying them all vary greatly, with phages of different virion morphology, size, and host-range all participants in an orgy of recombination [36]."

Hence the advice above. Just explain some version of the above to the reviewer, and let GenBank/ICTV apply whatever designations they see fit!

Posted in: General Message Board → Classificiation with ICTV guidelines

Link to this post \| posted 08 Mar, 2024 00:32
DanRussell	Way back in our "60 phage paper", we grouped phages into clusters based on > 50% nucleotide similarity. Of course, exactly what "50% nucleotide similarity" meant depended on which metrics in particular you looked at, so we used several different ones. You can read about them in the paper. More recently, we've moved to comparing genes rather than nucleotides, and have used ~35% Gene Content Similarity to put phages into clusters. Now, we're probably moving towards using a metric called Protein Equivalence Quotient to cluster phages. It's an ongoing process, and there is no "right" answer and phages will always challenge us by not neatly fitting into bins. PhamClust paper There have been a few groups who have looked at geography, and though there doesn't seem to be any high-level correlation between geography and type of phages found, some subclusters or even genes have only been found in certain geographical areas. –Dan

Link to this post | posted 08 Mar, 2024 00:32

DanRussell

Way back in our "60 phage paper", we grouped phages into clusters based on > 50% nucleotide similarity. Of course, exactly what "50% nucleotide similarity" meant depended on which metrics in particular you looked at, so we used several different ones. You can read about them in the paper.

More recently, we've moved to comparing genes rather than nucleotides, and have used ~35% Gene Content Similarity to put phages into clusters.

Now, we're probably moving towards using a metric called Protein Equivalence Quotient to cluster phages. It's an ongoing process, and there is no "right" answer and phages will always challenge us by not neatly fitting into bins.
PhamClust paper

There have been a few groups who have looked at geography, and though there doesn't seem to be any high-level correlation between geography and type of phages found, some subclusters or even genes have only been found in certain geographical areas.

–Dan

Posted in: Phage Biology → Phage Clustering

Link to this post \| posted 13 Feb, 2024 14:57
DanRussell	eagodin Thanks for the update, Debbie! Does this mean that we shouldn't use Atuin, Mimi, or Racecar as comparators at all, since the sequences were incorrect? I think my students are up for a good challenge, but will certainly reach out with any questions that come up. Liz Hi Liz, Just to be clear, the sequences were correct in the sense that all the bases were right. The change was just where Base 1 was, and duplicating a portion of the genome. (Not changing any individual bases or anything.) So everything should be fine/comparable, including the original annotations of those complete ones, but numbers will have changed. –Dan

Link to this post | posted 13 Feb, 2024 14:57

DanRussell

eagodin
Thanks for the update, Debbie! Does this mean that we shouldn't use Atuin, Mimi, or Racecar as comparators at all, since the sequences were incorrect? I think my students are up for a good challenge, but will certainly reach out with any questions that come up.
Liz

Hi Liz,

Just to be clear, the sequences were correct in the sense that all the bases were right. The change was just where Base 1 was, and duplicating a portion of the genome. (Not changing any individual bases or anything.) So everything should be fine/comparable, including the original annotations of those complete ones, but numbers will have changed.

–Dan

Posted in: Phamerator → Cluster FC in Phamerator

Link to this post \| posted 08 Feb, 2024 19:31
DanRussell	Hi Nancy, Can you post the file here perhaps? Thanks, –Dan

Posted in: DNA Master → Error when opening FASTA file

Link to this post \| posted 10 Jan, 2024 21:38
DanRussell	jcaoyao@gmail.com Most thankful for your wonderful explanation, Dan! It does make sense. So having a Class 2 Biosafety Cabinet wouldn't make your lab a BSL2, and therefore, allow you to handle M. abscessus? Right, it's definitely more complicated than that! (At least for us at Pitt.)

Posted in: Mycobacterium → Legal statement

Link to this post \| posted 10 Jan, 2024 21:18
DanRussell	Hi Juan Carlos, Here in the Hatfull Lab at the University of Pittsburgh, all of our clinical M. abscessus isolates are classified as BSL-2, and for good reason. Sometimes an apparently "healthy human" may have undiagnosed or non-obvious issues, and treating an opportunistic pathogen as "harmless" is dangerous. As for the "ultimate, legal, foolproof" statement, that probably doesn't exist. BSL guidelines are set by your institution, and we at Pitt follow guidelines set by Pitt, the CDC, NIH, strain collections, etc. Different schools, countries, or organizations may have different rules, but we always expect all SEA-PHAGES schools to abide by appropriate BSL regulations in their region. We've had real safety concerns recently, as some SEA-PHAGES schools have tried phagehunting on bacteria they've isolated from the environment. While this offer a different phage yield, allowing students to work with an unknown bacterium and treating it as BSL-1 is not endorsed by the SEA-PHAGES program! –Dan Edited 10 Jan, 2024 21:37

Link to this post | posted 10 Jan, 2024 21:18

DanRussell

Hi Juan Carlos,

Here in the Hatfull Lab at the University of Pittsburgh, all of our clinical M. abscessus isolates are classified as BSL-2, and for good reason. Sometimes an apparently "healthy human" may have undiagnosed or non-obvious issues, and treating an opportunistic pathogen as "harmless" is dangerous.

As for the "ultimate, legal, foolproof" statement, that probably doesn't exist. BSL guidelines are set by your institution, and we at Pitt follow guidelines set by Pitt, the CDC, NIH, strain collections, etc. Different schools, countries, or organizations may have different rules, but we always expect all SEA-PHAGES schools to abide by appropriate BSL regulations in their region.

We've had real safety concerns recently, as some SEA-PHAGES schools have tried phagehunting on bacteria they've isolated from the environment. While this offer a different phage yield, allowing students to work with an unknown bacterium and treating it as BSL-1 is not endorsed by the SEA-PHAGES program!

–Dan

Edited 10 Jan, 2024 21:37

Posted in: Mycobacterium → Legal statement

Link to this post \| posted 03 Nov, 2023 16:47
DanRussell	Hi SEA-PHAGES faculty folks, We got the following message and wanted to pass it on to you in case you're looking for potential summer employment. "The Summer Science Program recently designed a program in bacterial genomics where participants evolve a BSL1 bacteria (V. natriegens) to tolerate ever-increasing levels of antibiotics. After the evolution and Illumina sequencing our participants assemble the genome and try to identify putative beneficial mutations. We are looking for faculty to lead the micro and the bioinformatics sides, and Phage Hunters seemed like a good place to find faculty." Details on their employment page: https://summerscience.org/employment/ –Dan

Link to this post | posted 03 Nov, 2023 16:47

DanRussell

Hi SEA-PHAGES faculty folks,

We got the following message and wanted to pass it on to you in case you're looking for potential summer employment.

"The Summer Science Program recently designed a program in bacterial genomics where participants evolve a BSL1 bacteria (V. natriegens) to tolerate ever-increasing levels of antibiotics. After the evolution and Illumina sequencing our participants assemble the genome and try to identify putative beneficial mutations. We are looking for faculty to lead the micro and the bioinformatics sides, and Phage Hunters seemed like a good place to find faculty."

Details on their employment page:
https://summerscience.org/employment/

–Dan

Posted in: General Message Board → Summer Faculty Job Opportunity

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