cnobles
commented
3 years ago Hi Liang,
The error is occurring due to the page being down. In my experience this is only a temporary issue. I would recommend just trying the link separate from the processing, and when you can get to the file through a browser, then try to finish your processing. I will reach out to the Bushman Lab a see if there is an issue.
In the future, you can download the files and have them within the iGUIDE directory (or your working directory) and provide a path to the file rather than a web link. Additionally, here is a proper format (only chose one format per file) to include in the config file should you want to change from web links to local files (you'll need the file first to do the local version):
# Web address format
oncoGeneList :
file : "http://bushmanlab.org/assets/doc/allOnco_Feb2017.tsv"
symbolCol : "symbol"
# Local file format
oncoGeneList :
file : "etc/allOnco_Feb2017.tsv"
symbolCol : "symbol"Regarding your question about the dsODN concentration, this is actually something I would highly recommend you try to optimize in the lab prior to sequencing. This can save you a lot of time and money. I would recommend doing a few conditions and then isolating genomic DNA. Design primers for your on-target side and then use the genomic material as template. The dsODN has a restriction site in the middle (...) so if you have solid incorporation of the oligo in the the genome, you should be able to digest the PCR amplicon and resolve this on a DNA gel. You can use this process to optimize your reaction conditions for when you have the desired amount of dsODN incorporated. Under those conditions, you will likely want to run your assay. Not everyone uses the same protocol for gene editing, as you've pointed out, so taking a step back and optimizing the protocol for your own use can be useful.
This last point might actually be able to address your second additional question. If the cluster generation is poor, you may have low yield in your libraries due to an inefficient amount of starting template. Additionally, the R2 read has a challenge of sequencing through a constant sequence region (super low diversity) which heavily impacts the output. I recommend checking which levels of phiX may be helpful, we frequently used 5-10% for the manuscript samples sequenced on a MiSeq. I suspect greater challenges for diversity if sequencing on a 2-color chemistry platform, such as the NextSeq.
Best, Chris
liliang0908
Hi Chris,
I met a problem when running iguide, the eval.log(error) as follows:
iGUIDE Evaluation Inputs: Loading dependencies. Importing experimental data and configurations. Error in download.file(input, tmpFile <- tempfile(), method = method, : cannot open URL 'http://bushmanlab.org/assets/doc/allOnco_Feb2017.tsv' Calls: suppressMessages ... withCallingHandlers -> loadRefFiles -> -> download.file
In addition: Warning message:
In download.file(input, tmpFile <- tempfile(), method = method, :
URL 'http://bushmanlab.org/assets/doc/allOnco_Feb2017.tsv': status was 'Couldn't resolve host name'
Execution halted
So it is likely that the URL 'http://bushmanlab.org/assets/doc/allOnco_Feb2017.tsv cant be connected, how to deal with it?
By the way, I have another two question about iGUIDE: 1 Article mentioned that:” deliver 100 to 500 pmols of dsODN along with the protein”, if i use 3*10^6 cell in 20 µl system with PNP, what is the recommand pmol of dsODN? 2 Cluster generation of my iGUIDE library is poor when sequencing, the quality of read2 is bad(sequence of read2 sequencing primer is provided in the iGUIDE article), do you have any suggestions for this problem?
You reply will be greatly appreciated!
best, Liang