why I get "Running twoBitToFa failed" when loading chopchop

Question

[WYZ1120zzh]

Hi everyone,

I'm working on chopchop to design sgRNAs for CRISPR/Cas9 on HPCC.

As to twoBitToFa, I could successfully use it(with path) to convert .2bit to .fasta. I tried sc, hg38 and K.p(reported). However, it turned out Running twoBitToFa failed loading chopchop. Does anyone know why and how to fix it?

I mainly followed instructions as follows.

• https://bitbucket.org/valenlab/chopchop/src/master/
• https://github.com/malumbreslab/chopchop
• https://github.com/ianwheeldon/Kphaffii_library_design

The previous steps I took are listed in the following:

1. get gene_table
2. download .2bit file
3. convert .2bit to .fasta using twoBitToFa
4. get compressed version using bowtie build
5. modify config.json

[Eric]

Did you get this fixed?

[GenoMax]

Can you clarify what step you are getting the error at and the command you are running? Original poster was not clear about that either.

[Eric]

This is the command:

/chopchop_query.py \
  --genePred_file "/path/to/Zmays.gene_table" \
  -G "Zmays" \
  -o "${OUTPUT}" \
  --consensusUnion \
  --scoringMethod DOENCH_2016 \
  -t WHOLE

It fails in coordToFasta of chopchop.py when executing line 1155

./twoBitToFa -seq=Chr4 -start=234540973 -end=234541809 /path/to/crispr/2bit_folderNew/Zmays.2bit stdout 2> /path/to/output/twoBitToFa.err

[Eric]

My issue was I didn't have full paths in my JSON

  "PATH": {
    "PRIMER3": "/path/to/chopchop/primer3_core",
    "BOWTIE": "/path/to/chopchop/bowtie/bowtie",
    "TWOBITTOFA": "/path/to/chopchop/twoBitToFa",

I can't speak for the original poster as they had full paths. I realized when I attempted to run the ./twoBitToFa -seq=Chr4... command manually that it worked as expected. Knowing it works I updated to full path and I have past this error.

[GenoMax]

Good to know.