CRISPRnano2

test

Optional parameters

Click to expand

Sequence (if genename given, leave it blank):

(? Click here!) Reference amplicon length should be in the range of 200-300 bases with the nuclease target site located roughly in the middle. You can get the reference from Ensembl link below. Click again to close

Interested region (optional) +/- bp:

(? Click here!) 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 value 0 is double strand breaking point- Crisprnano will use the first index of sgRNA mapped on reference. Click again to close

Indel Threshold [%]:

(? Click here!) The Indel Threshold 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 to use 5% for ONT reads and 2% for Illumina reads (default is set to 5%). Click again to close.

PHred Threshold:

(? Click here!) The Quality threshold determine the minimum filter base, For Illumina it should be >20, For ONT it should be >10, Click again to close.

dsDNA without Homology Arm

(? Click here!) The Quality threshold determine the minimum filter base, For Illumina it should be >20, For ONT it should be >10, Click again to close.

ssDNA with Homology Arm

(? Click here!) The Quality threshold determine the minimum filter base, For Illumina it should be >20, For ONT it should be >10, Click again to close.

Input data

FASTQ Files (*):

(? Click here!) FASTQ output files from Illumina or ONT. We recommend file's size smaller than 200MB. 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. Click again to close.

Data type:
Window size & indel rate will automatically adjusted

Gene Name (**):

(? Click here!) Name of the study gene (Human only). Click to close.

Gene names are from this list

Nuclease Target Site (*):

(? Click here!) In this field the nuclease target site is entered. The sequence has to be in the same orientation as the reference sequence above. Click again to close

Targeting mutagenesis oligonucleotide (Knock-in):

(? Click here!) Donor oligonucleotide sequence used to introduce a site specific genomic modification. The sequence entered should be in the same orientation as the amplicon sequence. Click again to close.

Prime Editing/Knockin-only mode (exclude INDEL)

CRISPRnano website is free and open to all users and there is no login required.
To use CRISPRnano, you must enable JavaScript on your web browser.
If you want to use CRISPRnano offline, please download the offline html file here.

(*) Mandatory field
(**) If Gene name is blank, customer sequence must given

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

SECONDARY ANALYSIS

SWITCH TO BULK/SINGLE CLONE VIEW

NOTE: Click to the sample after selecting above option. Bulk view use lower percentage threshold

OFF TARGET Analysis (optional)

List of off-target Gene names (separate by ,):

(? Click here!) Name of the study genes, seperate by , . Click to close.

Off-target analysis will check up to 4 genes in a single run

If off-target sgRNA empty, system will take the previous sgRNA to find region of interest

sgRNA gene 1

sgRNA gene 2

sgRNA gene 3

sgRNA gene 4

Manual

Source code on Github

Optional parameters

Input data

CRISPRnano manual

TEST DATA

SECONDARY ANALYSIS

OFF TARGET Analysis (optional)

Save data