I hope this letter finds you well. My name is Xiqiong Wang, a candidate Ph.D. student of China Agricultural University. And I am writing to ask for help about the application of LongAmpseq method when identifying large deletion in Crispr/Cas9 knockout experiment. I have tried my own raw data obtained by Illumina platform, but it doesn't work. The amplicon size is 936bp, and I designed two gRNAs to knockout 175 bp, an intermediate deletion. When I run the code, I tried the cut cite of one gRNA. It failed. When I used the data SRR16953483 and SRR16953486 and the LongAmpseq code from the published paper titled "Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing”，then same error occurs again. I don't know how to fix it. Please help me.
Dear Pan and Cao,
I hope this letter finds you well. My name is Xiqiong Wang, a candidate Ph.D. student of China Agricultural University. And I am writing to ask for help about the application of LongAmpseq method when identifying large deletion in Crispr/Cas9 knockout experiment. I have tried my own raw data obtained by Illumina platform, but it doesn't work. The amplicon size is 936bp, and I designed two gRNAs to knockout 175 bp, an intermediate deletion. When I run the code, I tried the cut cite of one gRNA. It failed. When I used the data SRR16953483 and SRR16953486 and the LongAmpseq code from the published paper titled "Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing”，then same error occurs again. I don't know how to fix it. Please help me.
This is my code of SRR16953486:
wget https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR16953486/SRR16953486
fastq-dump SRR16953486.sra --split-3 -O outdir # then I copy and rename the two fastq file as R1_001.fastq and R2_001.fastq into Illumina folder.
And please see the detailed code and error in the attached file named "error.txt". I downloaded the CRCh37 verion of human reference genome ( https://ftp.ensembl.org/pub/grch37/current/fasta/homo_sapiens/dna/Homo_sapiens.GRCh37.dna.primary_assembly.fa.gz ).
I would appreicate it if you could help me solve the problem.
Thank you very much!
Have a good day!
Sincerely,
Xiqiong Wang error.txt