Setting Guppy version

[vladimirsouza]

Hi, I want to use Pepper-Margin-DeepVariant with Singularity to call variants from nanopore sequencing data. The basecalling was done using Guppy 4.2.2, r9.4.1, hac. But when I use --help to see the options, I get only --ont_r9_guppy5_sup, --ont_r10_q20, or --hifi. Is there something like --ont_r9_guppy4_hac? If so, how can I download and use it?

In the absence of what I want, I tried --ont_r9_guppy5_sup. But I got the error cp: cannot stat '/opt/pepper_models/PEPPER_VARIANT_ONT_R941_GUPPY5_SUP_V8.pkl': No such file or directory. Do I have to download this file and save it in my /opt/pepper_models/, or is this something that is missing in the container image? Thanks for reading my message. I'm eager to see your answer. Bests.

[kishwarshafin]

hi @vladimirsouza ,

--ont_r9_guppy5_sup should work with hac reads. Can you please check if you are using the correct version which is r0.8

[vladimirsouza]

Thank you for your answer. Good to know that it works for hac. Does it work with Guppy 4 as well? Yes, I'm using r0.8. How can I solve the cp: cannot stat '/opt/pepper_models/PEPPER_VARIANT_ONT_R941_GUPPY5_SUP_V8.pkl': No such file or directory problem?

[kishwarshafin]

@vladimirsouza ,

If you are using guppy 4 then the suggestion would be to an older version of the software: https://github.com/kishwarshafin/pepper/blob/r0.4/docs/pipeline_docker/ONT_variant_calling.md

To resolve that issue, can you please put down the full command here so I can see if I can locally re-create the issue? There's also a case-study that you can follow here: https://github.com/kishwarshafin/pepper/blob/r0.8/docs/pipeline_singularity/ONT_variant_calling_singularity.md

[vladimirsouza]

Here is the code that I used:

REF=/home/vbarbo/project_2021/paper_analysis/reference/genome/GRCh38.p13_all_chr.fasta
BAM_DIR=/home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/data_manipulation
OUTPUT_DIR=/home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant
THREADS=20

mkdir -p ${OUTPUT_DIR}/pmdv
singularity exec --bind $OUTPUT_DIR/pmdv/,/usr/lib/locale/ \
  /home/mark/scratch/pepper_deepvariant-r0.8.simg \
  run_pepper_margin_deepvariant call_variant \
  -b ${BAM_DIR}/aln_s.bam \
  -f ${REF} \
  -o ${OUTPUT_DIR}/pmdv \
  -p pepperMarginDeepvariant_calls \
  -t ${THREADS} \
  --ont_r9_guppy5_sup

And here are all output message that I get:

[08-24-2022 06:49:44] INFO: VARIANT CALLING MODULE SELECTED
[08-24-2022 06:49:44] INFO: [1/9] RUNNING THE FOLLOWING COMMAND
-------
mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv;
mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/logs;
mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/intermediate_files;
cp /opt/pepper_models/PEPPER_VARIANT_ONT_R941_GUPPY5_SUP_V8.pkl /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/intermediate_files
-------
cp: cannot stat '/opt/pepper_models/PEPPER_VARIANT_ONT_R941_GUPPY5_SUP_V8.pkl': No such file or directory
[08-24-2022 06:49:44] ERROR: None]
[08-24-2022 06:49:44] THE FOLLOWING COMMAND FAILED: mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv;
mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/logs;
mkdir -p /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/intermediate_files;
cp /opt/pepper_models/PEPPER_VARIANT_ONT_R941_GUPPY5_SUP_V8.pkl /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv/intermediate_files]

P.S.: I have permition to write in directory /home/vbarbo/project_2021/paper_analysis/rebuttal/nanopore/files/ENCFF263YFG/variant_calling_from_nanopore/pepperMarginDeepvariant/pmdv.

[vladimirsouza]

kishwarshafin, Thank you very much. As suggested here, I switched to the older version pepper_deepvariant:r0.4, and it looks like the analysis is going all right with my Guppy4-generated sequences now. Bests.