Too few mapped reads (Cannot find guide)?

[hittsuji]

I tried to run CRISPResso2 (CRISPRessoWGS) on my nanopore sequencing reads using sorted bam file using minimap2 and samtoold (on macOS15.2 on intel Mac), but it did not give me any results.
The log showing:

CRISPResso version 2.3.2
[Command used]:
/opt/anaconda3/envs/crispresso2_env/bin/CRISPRessoWGS -b ../cact_inj_s.bam -f ./descr_cact.txt -r ../Cact.fasta -o CRISPRessoWGS_Cact_inj2

[Execution log]:
Index file for input .bam file exists, skipping generation.
Indexing reference file... Please be patient!
Retrieving reference sequences for amplicons and checking for sgRNAs
Cannot find guide TCGCAAGTGTTTTCCGAGCG in amplicon 0 (chr_id Cact
bpstart 825
bpend 1025
sgRNA TCGCAAGTGTTTTCCGAGCG
Expected_HDR NaN
Coding_sequence NaN
sequence
Name: gCact1, dtype: object)

Processing each region...
Running CRISPResso on each region...

The region [gCact1] has too few reads mapped to it (0)! Not running CRISPResso!
Skipping folder '/Users/hitoshitsujimoto/My Drive/My work/4_TAMU/Results/Sequencing/Plasmidsaurus/2025-01_U6/CRISPResso/CRISPRessoWGS_Cact_inj2/CRISPRessoWGS_on_cact_inj_s/CRISPResso_on_gCact1'. Cannot find run data status file at '/Users/hitoshitsujimoto/My Drive/My work/4_TAMU/Results/Sequencing/Plasmidsaurus/2025-01_U6/CRISPResso/CRISPRessoWGS_Cact_inj2/CRISPRessoWGS_on_cact_inj_s/CRISPResso_on_gCact1/CRISPResso2_info.json'.
Skipping the folder CRISPResso_on_gCact1: not enough reads, incomplete, or empty folder.
Analysis Complete!

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Even though it states that "Cannot find guide XXXX, the sequence present in my fasta file (Cact_ref.fa)

Cact
GGTATGTGGCAATGTGTGCGAGCGCAATGTTTGCAAACGGCGCTTCAATCTTGGCCTTTTAAAGAAATTTTAAGGTTTCGTTCATCAAATCGGCCTTTTTCGTATACTTCATCTGAACATACAGAAAAGTGAGCTTTTTATTTTGATTCGATGATTTTGCACTCTGATTTAATTTCTTCTCAATTTGTTGAGTTAAACATAGTTCACTGTATCCCTCAGAGTAAAGTTATTTTTTGAGTAATTTTTTCAATGCAAAAATGATTAACTGATAAACTTAACAGTGAAGAATTGAGACAATACTGAGAAGGAGGATATTATCAGAAATCCTAACGTTGTGGAGCCAATCGTGATAGTAATGGAAGGGATTTTACTTCTATTGTTGTTCTACCATATCTACTGAGAATGTGGAAAGAAACAGTTTTGATTTACTTATTCCACAAACCTGCAGCAGAGTCAAGTGGGCTTTAAACCATGTTTGCAAAGCAAATTGGCATTGCTTCACTGAAAATCCCAAACCACAGTGATTATGAAAGTTCGTCGCCATATGGCACGGGTTGTTGTTTACTCCTCTGCTGGGGAAATTGGAAGTCCCCTTCTGTATGATCCCCGTCAGCTGGTCGCACTAGGATCTCCCCATAATTTGACTGATTTTGTACGCGATGTGATTCGCTCTCGTGACGTAATAACGGTAATCGTAGTGTGTAGTTCTGTCGATTCGACAGTCAGTCAGATCTGACGATGATACACACAAACCCTATTTAGCAATGTGGGAATTACACACTATACAGAGAGCACAGTGTCCATAATGTAGTTTCCGTCCTCGCTGGCTTTGCATTCGCGCGCTCTCTGCTCAGCTCGGAGACATcgttcgccgttcgaagtcagttcggtttgagctgttgagcagatcgcaagtgttttccgagcgaggctttttgagtggttcacgtgtggtgagcgcattcgcgattttttttttgtataaaatctacccgcgacgttcgtgtgttcgcgcgtaccaacacatgctagggtacgtacttctgcattgagacgggtactcagctctagaagtgattaccgagcttcgggtgtgtgcgtgtgctggacaagtgctggtacctgattgtgtcagctcgttattttgtagattgaaaacttaaacactacatgcagtattggaggaattaatttacgagtggatagtgaacccgtaggtgccctatttagatttgacctttaacgtgatactgtactgaggttccgagtatacattgtaagttctttgctcgggtgaaaagtgcatacagaatatgtggagaacatctgtgtcagcgaaagtgacttatttgaatcgccatcttgagttaatgtgttgaataccgattagagaaaagagccatatctacattggccagtgtcaatagccattgcaggcctttaatcatcttgcgttgaagtgagtggttattgagcgaagatagtttgtcgagaggcttgccaaacgaacaaacaaaaaagatccagttcttttcaaacaagcagaccaacATGTATCCATCAAAAGGAGGAGCGTCGTCAGCTGGAGGTTTCACGCACCTTTCCCTTTCAGAGGGTCAAAATAAGCAGGACAAATTTTCCACCGACAGCGGAGCGATAGACTCGGGCTTCCATTCCGGGCAGAACTTCAGCTCGGACAATCTGTTCAGCTCGTCTTCGTCGGCGTCAGTGCAACCGGAAGAAGCTCCAGTGCAAGTGCTGTACCAATCCGGTAGCCAGAAGCAACCCCTGGAGGAAGACAGCCGCACCTTCGATTCCGGTGTGGATCTAGACTCGAGTCAGCAGAACCCGAGCAGCGCCGATCCGATGAAGGTGGACGACCTGTGCATCAGCCTGCGCCAAATGCAGATGAAGTCCAAGGAGCAACAACAGGCGCTGCATTTGATGAAATGGGAAAAGTACTTCCACCAGAACGACGAC

I am not sure wha's going on..

[kclem]

Hi @hittsuji,

Regarding the 'Cannot find guide' error, I'd double check that you're passing the same fasta file to CRISPResso. In your command you provide ../Cact.fasta while you are printing out Cact_ref.fa.

CRISPRessoWGS will only consider reads that completely align to your region files, so if your reads don't span this entire region, CRISPResso will report no reads aligned to this region.

Let me know if you have any other questions!

[hittsuji]

Hi kclem,
My bad. I pasted the wrong command. I wanted to check if I make the fasta file name same as the chromosome name (Cact) with the fasta file named "Cact.fasta". The same results either way.

Command with Cact_ref.fa was:
/opt/anaconda3/envs/crispresso2_env/bin/CRISPRessoWGS -b ../cact_inj_s.bam -f ./descr_cact.txt -r ../Cact_ref.fa -o CRISPRessoWGS_Cact_inj

[hittsuji]

I tried to run on a server computer (Linux, CentOS 7) via ssh. It also utilizes anaconda. And the command worked. I am still seeking to solve issues on my computer, but it may be platform specific.

[kclem]

I'm glad it works on the other computer!

If you find out what the problem was, feel free to post it here in case others encounter it

[hittsuji]

Please post this back on the discussion, because I don't know how to troubleshoot this on my computer. I would like to make it work on my computer.