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Reference:
Nguyen, T., Ramachandran, H., Martins, S., Krutmann, J. and Rossi, A., 2022. Identification of genome edited cells using CRISPRnano. Nucleic Acids Research.

Question: thach.nguyen**(replace with email symbol)**iuf-duesseldorf.de

About:

CRISPRnano is a javascript-based program that was developed for rapid deep sequencing based genotyping of nuclease edited cell clones. CRISPRnano supports noisy Oxford Nanopore but also Next Generation Sequencing (NGS) reads such as Illumina and PacBio or Sanger Sequencing.

CRISPRnano was developed by Dr. Thach Nguyen in the group of Dr. Andrea Rossi (GEMD lab) at Leibniz Research Institute for Environmental Medicine. The template web interface, data input, output are developed based on the template of Outknocker http://www.outknocker.org/.


Licence:

CRISPRnano can be redistributed and/or modified under the terms of the GNU General Public License (Version 2), as published by the Free Software Foundation. A copy of the license can be found online at www.gnu.org/licenses. CRISPRnano is distributed in the hope that it will be useful, but without any warranty; without even the implied warranty of merchantability or fitness for a particular purpose. See the GNU General Public License for more details.

Citing CRISPRnano:


We kindly request that use of this software be cited in publications as:
Nguyen, T., Ramachandran, H., Martins, S., Krutmann, J. and Rossi, A., 2022. Identification of genome edited cells using CRISPRnano. Nucleic Acids Research.

CRISPRnano manual

CRISPRnano is a javascript-based program that was developed for rapid deep sequencing based genotyping of nuclease edited cell clones. CRISPRnano supports noisy Oxford Nanopore but also Next Generation Sequencing (NGS) reads such as Illumina, PacBio, or classic Sanger Sequencing.

Input data:

CRISPRnano analyzes FASTQ output files from ONT, NGS, or Sanger systems. CRISPRnano webserver analyzes multiple samples and supports up to 96 FASTQ files at the time. We recommend a file's size smaller than 200MB. Please, make sure that the correct ordering is applied in the file browser. The file allocation to individual pie charts can be verified by the numbers given at alignment track. Our data contains multiple small FASTQ files. FASTQ files can be concatenated using command cat under Linux or type under Windows environment.
On Linux terminal:
$ cat /path/to/fastq/files/*.fastq > /your/new/location/output.fastq

On Window command prompt (path symbol is different):
$ type \path\to\fastq\files\*.fastq> \your\new\location\output.fastq

Reference sequences
Reference amplicon length should be in the range of 200-350 bases (based on your library preparation), with the nuclease target site located roughly in the middle. The reference sequence of your gene of interest can be retrieved from Ensembl database.

Nuclease Target Site
The Nuclease Target Site sequence (CRISPR, TALEN, ZINC-Finger, etc.) has to be in the same orientation as the reference sequence above. The Nuclease Target Site is briefly checked if it is contained in Reference genome

Interested region and Offset centre
For ONT reads, it is recommended to use -/+15 bases around the predicted DNA lesion. Larger regions are prone to false positive indels. Larger regions (total of 200-250 bp) can be safely used with Illumina reads. You can drag and drop the central point of the predicted double strand break in the genomic sequence to the left or to the right. Default 0 is double strand breaking point.

Targeting mutagenesis oligonucleotide
Donor oligonucleotide sequence is used to introduce a site specific genomic modification. The sequence entered should be in the same orientation as the amplicon sequence.

Indel Threshold
The Indel Threshold percentage determines at what relative occurrence an individual indel mutation is considered as a unique allele of the respective clone analyzed, and is thus displayed in the alignment list. Low threshold values can result in false positive allele calling due to sequencing errors, whereas high threshold values result in false negative allele calling. Values in between 0 - 100 % can be entered. We recommend using 5% for ONT reads and 2% for Illumina reads (the default is set to 5%).

Code Availability
The source code for CRISPRnano is available at https://github.com/thachnguyen/CRISPRnano

Test data
The test data for CRISPRnano is available at our test sample.

Browser compatibility

OS	Version	Chrome	Firefox	Microsoft Edge	Safari
Linux	Ubuntu 20.04	Version 122.0.6261.128	125.0	n/a	n/a
MacOS	Monterey	Version 122.0.6261.128	125.0	n/a	15.3
Windows	10	Version 122.0.6261.128	98.0	122.0.6261.128	n/a

TEST DATA

Code Availability
The source code for CRISPRnano is available at https://github.com/thachnguyen/CRISPRnano

Test data
The test data for CRISPRnano is available at our test sample.

Browser compatibility

OS	Version	Chrome	Firefox	Microsoft Edge	Safari
Linux	Ubuntu 20.04	Version 99.0.4844.51	98.0	n/a	n/a
MacOS	Monterey	Version 99.0.4844.51	98.0	n/a	15.3
Windows	10	Version 99.0.4844.51	98.0	99.0.115039	n/a