All posts by Andy
ICTV Bacteriophage Genera
With the influx of phage genomic data, there have been several changes to bacteriophage taxonomy. See the paper “A Roadmap for Genome-Based Phage Taxonomy” by Evelien Adriaenssens and Dann Turner who have led these efforts with their work with ICTV. As a result, the classical phage families of Podoviridae, Siphoviridae and Myoviridae are: kaput, have shuffled off their mortal coil, given up the ghost, or any other preferred phrase… generally, they are no more. Some will be upset by this, I am sure. But personally, I am more than happy to see them go. While they were useful for a period of time, the ability to rapidly sequence most phage genomes and put phages “in boxes” based on their genomic content, rather than what they looked like, offers so much resolution to describe differences. For Podoviridae, Siphoviridae and Myoviridae aficionados, the morphotypes of Myovirus, Podovirus and Siphovirus live on.
As a result of updates to phage taxonomy, 100s of new genera have now been created. For our own work, we are interested in rapidly identifying which genus newly isolated phages fall within. Consequently, we have collected genomes of all (dsDNA) phages classified by ICTV and organised them into directories based on genus. Extracted the common marker terL for each phage that can then be used for input into alignments for creating phylogenies, to determine how related a new phage may be. Additionally, we have run VIRIDIC on each genus.
We are hoping to have an automated process available that will rapidly identify if a new phage genome falls within a known phage genus, based on currently ICTV guidelines. As others are probably comparing phage genomes to know taxa, we have made all the data downloadable as a single file called ICTV_genera.tar.gz, which can be downloaded here as it may well be useful to others.
It contains 1653 directories (Genera)
Each directory contains:
- *gff files of every ICTV classified phage genome of that genus.
- *fsa individual fasta files every ICTV classified phage genome of that genus.
- *_terL.ffn – automated extract of terL gene of every ICTV classified phage genome of that genus.
- 04_VIRIDIC_out folder
- *pdf heatmap for that genus
- *clusters.csv file from VIRIDIC for that genus
- *MA_genCol.csv from VIRIDIC for that genus
- If genera only have 1 species, we don’t run VIRIDIC for obvious reasons
- If the directory is empty, it is a genus of an RNA phage (still sorting this- see above about dsDNA)
To get the data use :
wget http://warwick.s3.climb.ac.uk/inphared/ICTV_genera.tar.gz tar -xvf ICTV_genera.tar.gz
If you find this useful, consider citing Cook, et al 2021. INfrastructure for a PHAge REference Database: Identification of Large-Scale Biases in the Current Collection of Cultured Phage Genomes. PHAGE. https://doi.org/10.1089/phage.2021.0007
Tinkering with size selection for nanopore sequencing of viromes
Ryan has recently been testing the short read eliminator kit from circulomics to enrich for long read for nanopore sequencing of viromes. The input for virome samples was various liquids produced or excreted by cows ….often smelly and sticky, generally not much fun to extract the viral fraction from.
Given the nature of the samples getting large amounts of DNA, let alone HMW DNA from the small sample volumes is not possible. Resorting to MDA amplification to produce enough DNA for the nanopore sequencing. To enrich for longer reads Ryan tried multiple samples without any enrichment with the short read eliminator kit.
The results of which look encouraging
It shifts the median read length from 1.9 kb to 6.9 kb, which still aren’t huge reads. But given average size of phage genomes and the low input issues, it might make a difference to the final assembly. Initial results of assembly of a small number of samples look encouraging, with ~1000 predicted complete phage genomes as predicted from checkV. This is in line with similar number of genomes we have previously assembled from a single seawater sample, where we found a 650 kb phage genome.
We still have to determine how this SRE kit might exclude some phages that have small genomes. As we have previously observed differences in the population of smaller phage genomes when comparing nanopore and illumina sequencing (Cook et al 2021 Microbiome).
*As i found this recently- here is Ryan talking about some of his previous work on PromethION sequencing of viromes
PHAGE ANNOTATION WITH PHROGS
Recently PHROGs was released by Terzian et al (https://doi.org/10.1093/nargab/lqab067 ). Full details are provided on their webpages and publication. Briefly their curated dataset provides tens of thousands of PHROGs with a standardised annotation attributed to each PHROG. All of this is available through their searchable website and can also be downloaded.
For first pass phage genome annotation this seems like a great resources. We standardly use Prokka for annotation of phage genomes, that allows custom hmm databases to be used for annotation. Unfortunately the HMMs provided directly by the PHROGs team don`t sit neatly into Prokka and allow the annotation linked to the PHROG to appear in the final annotation, because of differences in formats.
However, as they provided all their data in an easily downloadable form. We have taken this and reformatted to produce HMMs with the annotations included so it plays nicely with HMMER3 as part of Prokka . We have produced a single file that can but put in /opt/prokka/db/hmm directory of Prokka. Thanks to Thomas Sicheritz-Pontén for helping with sorting out getting the correct annotation into the 38,000 HMMs …
A single file containing all HMMs that can be directly added to Prokka , can be downloaded here. Warning its 3 Gb when unzipped. Thanks to Terzian et al who did all the hard work on producing the original PHROGs and curated annotation and making it available , we have just reformatted it for our own use and anybody else that might want to use it with prokka..
To get it running within prokka. Locate the installation of prokka
In my case this results in output of /usr/local/bioinf/prokka/db
and [08:43:23] * HMMs: all_VOG HAMAP
telling us there are already some HMMs databases called all_VOG & HAMAP
Within /usr/local/bioinf/prokka/db is the a directory called hmm
Thus, the full path is /usr/local/bioinf/prokka/db/hmm
The downloaded database needs to be copied into /usr/local/bioinf/prokka/db/hmm
Then run $prokka –setupdb
Running the command $prokka –listdb
[08:43:23] * HMMs: all_phrogs all_VOG HAMAP
all_phrogs will now be used by prokka. If you only want to use the PHROGs database, consider using the prokka flag of –hmms and specify /usr/local/bioinf/prokka/db/hmm/all_phrogs
Full details on adding databases are explained on the Prokka github page
Bias in phage genomes
What started off sometime in 2019 as search for a number, too put into an introduction of a paper ends up a few years later with hopefully a useful paper. That number was how many complete phage genomes are currently publicly available via public databases are currently available. At the time, NCBI virus had not been released (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/), which contains some of this information. Myself and Nathan Brown wrote a quick script that used the esearch/efetch factilies to extract phage genomes. Then applied several filtering steps to extract “complete” phage genomes with lots of manual filtering. We started providing this data on the website for download. After requests from people of how to cite this list and some reminding from Branko Rihtman, we have finally got to a pre-print. Ryan Cook has tidied up the code a lot and parsed lots of informtion that can be extracted from the genbank files.
In extracting this informaiton we found many things
There is big bias in the hosts that phage are isolated on – most phages are isolated on a small number of host bacteria
Far more lytic phage genomes than temperate – with most temperate phage genomes coming from an even smaller number of hosts
The number of putative antibiotic resistance genes is different for lytic versus temperate phages and host
Jumbo phages are not always rare – again dependenent on the host
Even for hosts where large numbers of phage have been isolated, we are a long way from sampling the number predicted phage species t
All the data can be accessed via github https://github.com/RyanCook94/
And the paper on https://www.biorxiv.org/content/10.1101/2021.05.01.442102v1.article-metrics
All v all comparison of coliphages
Having recently sequenced several coliphages, we have wanted to compare them to all other coliphages. To do this, we have downloaded all complete (or near complete) bacteriophages genomes [see here]. We then filtered these genomes based on their GenBank description to pull out all phages that have Escherichia, E.coli or coliphage in their description. Having done this we then used an all v all comparison of using MASH, to construct a matrix of similarity. Then visualised this using the heatmaply.
This can be seen below. An interactive webpage of the image is available here
Looking closely at the clusters it is clear to see that phage with genus form discrete clusters eg top right of the plot is T4virus (and other genera in the Tevenvirinae subfamily)
We have moved ….
The lab has now moved from Warwick Medical school to the Dept of Infection, Immunity, and Inflammation at the University of Leicester. To be more specific, I have moved with the rest of lab group still at Warwick.
After 17 years at Warwick and knowing who to speak to and where to find things, it has been an interesting experience starting at Leicester. Not knowing how to get into or where exactly my office/lab is in the building, has provided a new experience. But also great to meet new colleagues, who have helped me find my way.
Welcome to Branko and Slawek
October has brought the start of a new term and the arrival of two new lab members (ok it November before posting) Branko joins the lab to work as an ESPRC fellow working on AMR, he joins Paul who has further extended his ESPRC fellowship and will be with us for a few more months.
Slawek joins as a CENTA PhD student, who will be looking at the role of the marine VIROME in the maintaining a pool of AMR genes.
*Alex Wilcox has also joined the lab and like Branko and Paul he has gained an ESPRC fellowship
Bacteriophage genome assembly and annotation workshop
We will be running a bacteriophage genome assembly and annotation workshop at WMS on Monday 9th of January. The course will be run on CLIMB virtual machines, so please register for an account in advance.
Date: Monday 9th January 2017
Registration and payment by Monday 5th December* – registration form is open – here
Attendees may provide up to four samples of bacteriophage DNA (by 5th Dec 2016) in advance of the workshop, which will be sequenced and the data available for analysis on the 9th of Jan. During the workshop attendees will learn how to quality control their data, assemble bacteriophage genomes, annotate and prepare their genome in a format for submission to EBI.
Registration is on a first come first served basis, so register early. Spaces are limited to 20 people
MRC CLIMB infrastructure will be used for the workshop.
No prior experience of genome annotation is needed.
Analysis will be run on CLIMB. Users are encouraged to bring their own laptop – a limited number of laptops are available.
A free CLIMB account is also needed. Register here
DNA samples must be received by Monday 5th December for them to be sequenced in time for the course. It is not necessary to send phage samples to attend the course. The workshop is a genome annotation training workshop- sequencing of isolates is a bonus so that attendees can annotate their own genomes.
DNA samples must be sent in a 96 well plate. A minimum of 10 ul of DNA at 10 ng /ul DNA is required. Larger volumes are fine, but must be at concentration of 10 ng / ul.
DNA must be column purified prior to sending, Zymo DNA purification/columns are recommended. Concentration must be determined by use of fluorescent detection system (eg Qubit), not Nanodrop .
Phage do not have to be CsCl purified prior to DNA extraction. The method we use for extraction can be found here https://peerj.com/articles/2055/ , then run through a DNA clean-up column
Please contact Andrew Millard on email@example.com after registration, prior to sending any samples. DO NOT JUST SEND SAMPLES
- Contact firstname.lastname@example.org prior to sending
- Label the plate so that your name can be read on the side of the plate.
- Complete the form that will be sent when you contact email@example.com
- Sample names are to be alphanumeric only.
It broke :-(
We lost all our content . But hopefully it will be back soon ….
And then Branko fixed it 🙂