SEA-PHAGES | All posts created by jcaoyao@gmail.com

Link to this post \| posted 17 Sep, 2023 17:02
jcaoyao@gmail.com	Thank you a million for all your support.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 08 Sep, 2023 11:33
jcaoyao@gmail.com	Thank you very much, Sir. Would you know why assembling for example 200,000 lines using that command takes Newbler forever to run? On most of my genomes, it will simply get stuck after hours of waiting, and I just had to shut down the program. Should my Newbler be upgraded?

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 05 Sep, 2023 20:58
jcaoyao@gmail.com	Much grateful, cdshaffer. My genomes were also sequenced in 2x150 paired-end fashion. I did assemble using R1 and R2 reads individually, which did not come out well. Since I have a PC, I guess Pear won't work either. The messy results I got were indeed after downsampling using the 'head -n 20000' command. I checked my 454LargeContigs.fna file, and the longest contig is 27916 bp and there are 6,441 reads for it. Upon BLASTn, a long list of hits came up, and all were of phage genomes. The highest hit was phage Zakhe101, whose genome is 69,653 bp.

Link to this post | posted 05 Sep, 2023 20:58

jcaoyao@gmail.com

Much grateful, cdshaffer. My genomes were also sequenced in 2x150 paired-end fashion. I did assemble using R1 and R2 reads individually, which did not come out well. Since I have a PC, I guess Pear won't work either. The messy results I got were indeed after downsampling using the 'head -n 20000' command. I checked my 454LargeContigs.fna file, and the longest contig is 27916 bp and there are 6,441 reads for it. Upon BLASTn, a long list of hits came up, and all were of phage genomes. The highest hit was phage Zakhe101, whose genome is 69,653 bp.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 05 Sep, 2023 10:49
jcaoyao@gmail.com	Hello Dear Dr Russell, The Illumina generates 2 fastq files per phage genome, called R1 and R2. Your Newbler assembly tutorials used only one fastq file to assemble the genome. Is that the R1 or R2 file? Should I use either R1 or R2 fastq files separately to assemble and see how both results corroborate each other? Or should I merge both R1 and R2 files into one fastq and assemble that? So far, each file when run individually, has given me 100+ contigs (a couple large ones plus around 100 short contigs of about 100 bps), despite having been downsampled. Could this be due to contamination during DNA extraction? Thank you a lot.

Link to this post | posted 05 Sep, 2023 10:49

jcaoyao@gmail.com

Hello Dear Dr Russell,

The Illumina generates 2 fastq files per phage genome, called R1 and R2. Your Newbler assembly tutorials used only one fastq file to assemble the genome. Is that the R1 or R2 file?

Should I use either R1 or R2 fastq files separately to assemble and see how both results corroborate each other? Or should I merge both R1 and R2 files into one fastq and assemble that? So far, each file when run individually, has given me 100+ contigs (a couple large ones plus around 100 short contigs of about 100 bps), despite having been downsampled. Could this be due to contamination during DNA extraction?

Thank you a lot.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 21 Jul, 2023 19:31
jcaoyao@gmail.com	Thank you Sir. My case is precisely seafaculty account and failing. Every day it says like sorry, try again. 275Kb

Posted in: Phamerator → Install Guest Additions to VM ---- Without "SEAFaculty Login ability"

Link to this post \| posted 21 Jul, 2023 10:19
jcaoyao@gmail.com	Dear Dan, would you have the password/s for the guest addition command or even the streamlining software package you wrote? They are most important for me.

Posted in: Phamerator → Install Guest Additions to VM ---- Without "SEAFaculty Login ability"

Link to this post \| posted 19 Jul, 2023 15:59
jcaoyao@gmail.com	DanRussell jcaoyao@gmail.com Hello Staff, I can't move files from my computer to the VM, so I tried clicking "Insert Guest Additions CD Image…", but nothing happened, no activity occurred in the terminal window or anywhere. What could I do, please? Thank you. Hi, you can try opening a terminal and running this command: `sudo apt-get install virtualbox-guest-utils` If that doesn't work, I'd suggest asking the internet! –Dan Thanks Dan, for it does work. Which now it asks me for the [sudo] password for seafaculty, but isn't the one you once gave me. What would the new password be?

Link to this post | posted 19 Jul, 2023 15:59

jcaoyao@gmail.com

DanRussell
jcaoyao@gmail.com
Hello Staff, I can't move files from my computer to the VM, so I tried clicking "Insert Guest Additions CD Image…", but nothing happened, no activity occurred in the terminal window or anywhere. What could I do, please? Thank you.

Hi, you can try opening a terminal and running this command:
sudo apt-get install virtualbox-guest-utils
If that doesn't work, I'd suggest asking the internet!

–Dan

Thanks Dan, for it does work. Which now it asks me for the [sudo] password for seafaculty, but isn't the one you once gave me. What would the new password be?

Posted in: Phamerator → Install Guest Additions to VM ---- Without "SEAFaculty Login ability"

Link to this post \| posted 19 Jul, 2023 10:22
jcaoyao@gmail.com	DanRussell Hi Kyle, Good questions! There are a few resources that might be helpful here. One is that I wrote a small software package that helps streamline some of the assembly/QC process for phage genomes. It's called phageAssembler and is on github. https://github.com/SEA-PHAGES/phageAssembler It's only really meant to be installed on the 2017 SEA Virtual Machine. (I didn't really spend the time to make it thoroughly cross-platform.) But it should work there if you follow the Quick Start instructions. Because Newbler and consed are already installed on the SEA VM, it can use those installations and basically does the following: INPUT: fastq file 1. Downsample reads from your fastq file to get a workable number (default 80,000) 2. Assemble those reads with Newbler 3. Report #s of contigs & sizes 4. BLAST large contigs against a phage database and report possible cluster 5. Attempt to locate base 1 by similarity to genomes in the database 6. Report coverage and GC% of assembled contigs 7. Run AceUtil to search and tag assembly weak areas 8. Create consed-ready file for review 9. Write findings to a log file You can certainly do all those steps independently if you'd like to learn the process, but this script kind of gets you to the actual analysis part, skipping a lot of the need to learn command-line stuff for many different programs. The second resource is a chapter I wrote that details the whole process: https://pubmed.ncbi.nlm.nih.gov/29134591/ (If you can't get access, I can share the manuscript.) It's a more general look at what things you need to think about when sequencing and finishing phage genomes. Finally, there are some video tutorials I made that walk through some of the assembly/finishing process. These are a bit old and potentially outdated, but probably still have some useful info if you want to do more of the steps yourself. https://phagesdb.org/workflow/Sequencing/ And also, if you do sequence/assemble your own, we would definitely like to double-check them and include them in PhagesDB. To do so, we'd need your final sequence file and the sequencing reads. Hope that helps! –Dan Hi Dan, I would love to use your script, but it asks for some passwords. May I ask what they are? Thanks very much.

Link to this post | posted 19 Jul, 2023 10:22

jcaoyao@gmail.com

DanRussell
Hi Kyle,

Good questions! There are a few resources that might be helpful here. One is that I wrote a small software package that helps streamline some of the assembly/QC process for phage genomes. It's called phageAssembler and is on github.

https://github.com/SEA-PHAGES/phageAssembler

It's only really meant to be installed on the 2017 SEA Virtual Machine. (I didn't really spend the time to make it thoroughly cross-platform.) But it should work there if you follow the Quick Start instructions. Because Newbler and consed are already installed on the SEA VM, it can use those installations and basically does the following:

INPUT: fastq file
1. Downsample reads from your fastq file to get a workable number (default 80,000)
2. Assemble those reads with Newbler
3. Report #s of contigs & sizes
4. BLAST large contigs against a phage database and report possible cluster
5. Attempt to locate base 1 by similarity to genomes in the database
6. Report coverage and GC% of assembled contigs
7. Run AceUtil to search and tag assembly weak areas
8. Create consed-ready file for review
9. Write findings to a log file

You can certainly do all those steps independently if you'd like to learn the process, but this script kind of gets you to the actual analysis part, skipping a lot of the need to learn command-line stuff for many different programs.

The second resource is a chapter I wrote that details the whole process:
https://pubmed.ncbi.nlm.nih.gov/29134591/

(If you can't get access, I can share the manuscript.) It's a more general look at what things you need to think about when sequencing and finishing phage genomes.

Finally, there are some video tutorials I made that walk through some of the assembly/finishing process. These are a bit old and potentially outdated, but probably still have some useful info if you want to do more of the steps yourself.
https://phagesdb.org/workflow/Sequencing/

And also, if you do sequence/assemble your own, we would definitely like to double-check them and include them in PhagesDB. To do so, we'd need your final sequence file and the sequencing reads.

Hope that helps!
–Dan

Hi Dan, I would love to use your script, but it asks for some passwords. May I ask what they are? Thanks very much.

Posted in: Newbler → Getting Started with Phage Assembly

Link to this post \| posted 19 Jul, 2023 10:05
jcaoyao@gmail.com	Hello Staff, I can't move files from my computer to the VM, so I tried clicking "Insert Guest Additions CD Image…", but nothing happened, no activity occurred in the terminal window or anywhere. What could I do, please? Thank you.

Posted in: Phamerator → Install Guest Additions to VM ---- Without "SEAFaculty Login ability"

Link to this post \| posted 17 Jul, 2023 21:15
jcaoyao@gmail.com	Thank you Dr Shaffer for your thorough clarifications.

Posted in: Newbler → Getting Started with Phage Assembly

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