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pinellolab / CRISPResso2

Analysis of deep sequencing data for rapid and intuitive interpretation of genome editing experiments
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Alignment error, please check your input #13

Closed StevenVB12 closed 5 years ago

StevenVB12 commented 5 years ago

Dear Luca,

I am encountering the below error when running the command:

CRISPResso -r1 /work/rpapa/sbelleghem/mutant_miSeq/fastq_trimmed/TL12_R1_paired.fastq.gz -r2 /work/rpapa/sbelleghem/mutant_miSeq/fastq_trimmed/TL12_R2_paired.fastq.gz --amplicon_seq ATTGGATCTTAAAAGCTTGGGCTAAGCTCATGTCGACGGTCAGTAATTAGCATTCCGCATATAGTTTACAAAGCATTGCCGTTGTAAATTATTGGAAACTATAATCTTGTGCAAAAACTTGTTTTTTTATAAATATTATAAAATATATTCGTACAGGATTGAAATATAAAAAAAACATATCAGCTGCGAATAAAATTAATAGAGAATAAAAAAATATACTTATATCACAGCGACATATTTATTTTATTCTCTATTTTATTCACATTATATTTTTACTCCATGCCAAATTGATAATAGAATATGAACCTGTAACAACAGTCCTTAAAAATCCAAAACGATTATTAAGTGGTTTAATATTTTTACATAACAACATCAAATAATTTAAATTATATCTATTTCTAGGTAATACAGACAGGTGCTCAACAGGCGGTTGAAGAGTGTCAATACCAATTCCGAAACAGCCGCTGGAACTGCAGCACTGTCGAAAACAGCACTGATATATTTGGAGGAGTACTTAAATTTAGTAAGTAAAAGTTAAATTTTTGATTTAAATTTGTAAATCCTTTTTAATTGACAACCTAAATACTTATTTTTATTTGGATATATTATATAAAAATGTTGGATGAGTTTGGATTCCACTTACTACTTGGCTTCTTGAGCACTAACTTTAAAAATATATAAATTCTATTTGGAAAACGAAAGAAATAAGATTTCAAATGATCTATAACTAACAATTTTTATTATGATAAACCACAAACAACTATACAAAACGATTTACACGTAAAATTAACATATTCTCAACATATTACACAAATAATACTACCGTTAACTCAAAATTGGCATATACATATAAATAAATCTTGAATCATAAAATTCATTTCCGCTCGGATTTCAAGTCAAAGTAAGTTGTAAATTCTCAAATAATTATCGGTTGCATACATCGGCAACTCTTCAAAGGACGTGTTAAGTG --max_paired_end_reads_overlap 150 --name TL12 --output_folder /work/rpapa/sbelleghem/mutant_miSeq/CRISPResso_out

############### INFO @ Wed, 05 Jun 2019 13:10:08: Finished reads; N_TOT_READS: 29195 N_COMPUTED_ALN: 0 N_CACHED_ALN: 0 N_COMPUTED_NOTALN: 6874 N_CACHED_NOTALN: 22321

INFO @ Wed, 05 Jun 2019 13:10:08: Done!

INFO @ Wed, 05 Jun 2019 13:10:08: Quantifying indels/substitutions...

INFO @ Wed, 05 Jun 2019 13:10:08: Done!

CRITICAL @ Wed, 05 Jun 2019 13:10:08: Alignment error, please check your input.

ERROR: Error: No alignments were found #############

Would you have any advice on what to check to know what is going wrong? The amplicon should be fine as I can easily find alignable sequences in my fastq files.

Thank you for any help!

Steven

kclem commented 5 years ago

Hi Steven,

Thanks for using CRISPResso2.

It appears that your amplicon is 970bp long. CRISPResso2 is designed to analyze sequencing where each read completely covers the amplicon. Are your paired-end reads 500bp?

If your reads don't completely cover the amplicon, you should use CRISPRessoWGS.

  1. Provide the 970bp amplicon as your genome reference sequence(e.g. amp.fa)
  2. Align the reads to amp.fa with bwa or a similar aligner
  3. Create a description file with 'regions' of ~40bp around your cut site like this: chrAmp 20 60 Region1 CTACAGAGCCCCAGTCCTGG NA NA chrAmp 500 540 Region2 NA NA NA

Thanks, and let me know if you have any more questions.

Thanks,

Kendell

StevenVB12 commented 5 years ago

Thank you Luca,

I was able to make an exact amplicon and it worked!

Best regards,

Steven