Low number of reads aligned

[Frances-24]

Hi,
I performed a CRISPR knockout on an IPSC line using two guides. In most cases, I am getting an alignment of less than 50% of the reads. I think that this is probably due to a large deletion. Below, I provide an example (and I attach the fastq.gz file). Is there a way to use CRISPResso to see if I got a deletion in the reads?

Amplicon without edition:
AGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGG
GGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGCTCCACCTAGCCTCCCTGCAGCTGGGACT
GCTGTTAAACGGGGTCTGCATGGCTGAGGAGCTGCGCCACATCCACTCCAGGTGACTCACTGCAGTA
CCCAGGGACGGGGTATCCAAC

sgRNA:
AGGTACCGGAGAGTGTGCTC, GCTGGGACTGCTGTTAAACG

1a.Read_barplot.pdf

APOE44-STINGII-CR-clone-1-DB-FR-NIM_S80_L001_R1_001.fastq.gz

Thanks for your response,
Frances

[Colelyman]

Hi Frances,

Thanks for using CRISPResso! If you suspect you have a large deletion, I bet that reads are not meeting the default 60% homology cutoff. I would recommend setting the --default_min_aln_score parameter to 0, then every read that passes preprocessing will be aligned.

Let us know if this works for you!

Thanks,
Cole

[Frances-24]

Thanks a lot Colelyman for your quick reply. I will try that!

Frances

[Frances-24]

Hi Colelyman,

I don't find where I can change the cut off to 0. In"options" I find 50, 60, 70, 80, 90 and 100%:
.

Thanks

Frances

[Colelyman]

Sorry about that, I didn't realize you are using the web version. You can only set the score that low through the command line version. I have run the following command on your data and the results are attached (note that 100% of the reads are now aligned).

I didn't look too closely, but I would make sure that your amplicon sequence is correct (all of the primers removed, etc.).

CRISPResso -n frances-24 -a AGCCTTGGTTCTGCTGAGTGCCTGCCTGGTGACCCTTTGGGGGCTAGGAGAGCCACCAGAGCACACTCTCCGGTACCTGGCTCCACCTAGCCTCCCTGCAGCTGGGACTGCTGTTAAACGGGGTCTGCATGGCTGAGGAGCTGCGCCACATCCACTCCAGGTGACTCACTGCAGTACCCAGGGACGGGGTATCCAAC -g AGGTACCGGAGAGTGTGCTC,GCTGGGACTGCTGTTAAACG --default_min_aln_score 0 -r1 APOE44-STINGII-CR-clone-1-DB-FR-NIM_S80_L001_R1_001.fastq.gz --zip_output --place_report_in_output_folder

CRISPResso_on_frances-24.zip

[Frances-24]

Thanks Cole for your reply. I will ask for help to use the command line version in the bioinformatics core. The primers were not removed. So I would take this into account.