Apr 15, 2024
Version 2
Library Aligner and NGS Cas9 Cutting Analysis V.2
- 1FIVE@MGI;
- 2Washington University in St. Louis
- WashU FIVE @ MGI
Protocol Citation: Colin Kremitzki, William Buchser 2024. Library Aligner and NGS Cas9 Cutting Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3n89v4o/v2Version created by Colin Kremitzki
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 15, 2024
Last Modified: April 15, 2024
Protocol Integer ID: 98201
Keywords: ngs cas9 cutting analysis protocol, library aligner in fivtool, cas9 cutting analysis, library aligner, sequencing core, sequencing data, fivtool, grna library, analysis protocol
Disclaimer
This protocol will be adjusted to the best of our ability when FIVTools is updated, but it may not match exactly.
Abstract
Protocol on how to run Library Aligner in FIVTools once sequencing data has been returned to us from the Sequencing Core. It also has an option for checking the Representation of a gRNA library. Lastly, a video is posted on how to check your NGS reads for Cas9 cutting analysis.
Open FIVTools and click on the LA tab in the upper left section of the program.
Enter in your returned NGS link into the HTCF Link tab and the destination to download the files into Dest.
Press Download UZ to download the files and automatically unzip them. This will deposit reads into your destination folder inside folders called FastQ and FastQ_gz.
Once downloaded, you can press the button Defaults under XML Settings: to return the left criteria back to the defaults.
In the left tab for PathReferenceList, enter in the pathway which contains the reference .txt file. This file should contain the gRNA sequences that you wish to align to your reads. If you wish to align to multiple different .txt files, just list the directory and not a specific file.
In the left tab for PathFastQToSearch, enter in the pathway to your FastQ files.
The rest of the settings can be adjusted to fit your schema.
Press Start once you are ready to align your reads to your reference sequence list.
When LA is running, you will see several things happen on your screen. You will want to be looking for the % of Unmapped Reads. If this is 100%, then your reads are not matching anything in your reference list. If you have a low % of Unmapped Reads, then that means they are hitting to reference reads, and that’s a good thing.
Once finished, LA will deposit a lot of files in your destination folder. FastA files, ref_f.txt (what most people will want to look at - includes a counts table), ref_long.txt (fancier version used in Spotfire analysis), ref_TopSeq.txt (good for looking at your top hits if you get a low number of matches - answers the question: what are you matching to?).
If you wish to check the Representation of the gRNA libraries you’ve put into cells, you can do this at the bottom of Library Aligner.
Enter in the MOI you used to infect your cell line, the titer, and the Cell Factor. Cell Factor is 1 if you were using hearty cells like U2OS or HeLa cells. Cell Factor will be 2 if you were using delicate cells like iPSC cells. Select Merge P if you wish to merge SubPools together in the Representation Check, ie. MSPH1 through MSPH7 will be joined together. Press Check Rep when ready.
You can also check your representation via the following website: https://libalignerminiature20221013181154.azurewebsites.net/
Willie also created a youtube video on how to utilize FIVTools to check for Cas9 transfected gRNA cutting. That video can be found here: https://www.youtube.com/watch?v=b2_2suSgahg&list=PLHBFVqDRJN5VuV9CPXAfiiwMpZVOquSx_&index=35