guppy2sam produced an empty BAM file

[zhanxw]

Describe the bug guppy2sam produced an empty BAM file

Logging The relevant command lines are pasted:

(medaka) [xzhan9@Nucleus005 nanopore]$ medaka methylation guppy2sam --reference ${REFERENCE} ${FAST5PATH} \

--workers 32 --recursive \
| samtools sort -@ 8 | samtools view -b -@ 8 > ${OUTBAM}

[05:09:48 - ModExtract] NOTE: Mod. base scores are output w.r.t the sequencing direction, not the aligned read orientation. [05:09:48 - Extractor] Starting worker processes. [05:09:48 - Extractor] Found 81 files to process. [05:09:48 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_2.fast5. [05:09:48 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_67.fast5. [05:09:48 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_9.fast5. [05:09:48 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_27.fast5. [05:09:49 - Extractor] Extracted 1/81 files. [05:09:49 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_17.fast5. [05:09:49 - Extractor] Extracted 2/81 files. [05:09:49 - ModExtract] Processing fast5_pass/FAL15598_359b3b9b63235513017a3ad8c726078fdd3febd6_48.fast5. ...

Environment (if you do not have a GPU, write No GPU):

• Installation method: conda
• OS: Red Hat Enterprise Linux Server release 7.4 (Maipo)
• GPU model: no GPU
• Nvidia driver version: n/a
• CUDA version: n/a
• cuDNN version: n/a

Additional context The problem of this issue is similar to issue #129, but I don't think it is the same reason. I verified ont-fast5-api version is 2.0.1. For other versions, please see below:

ont-fast5-api==2.0.1 ont-tombo==1.5.1 medaka==0.11.5 samtools 1.9 (htslib 1.9)

The reference is GCF_000006765.1_ASM676v1_genomic.fna

[mwykes]

Hi @zhanxw, without having the data, it's quite hard to debug what is going wrong. Is it possible for you to share one of your fast5 files?

[cjw85]

@zhanxw

Can you confirm that when running guppy you are consistently using the modified base model, so you are running something like:

guppy_basecaller \
    --save_path <output path> --input_path <input path> \
    --compress_fastq --fast5_out \
    --config dna_r9.4.1_450bps_modbases_dam-dcm-cpg_hac_prom.cfg

[zhanxw]

I did not use this config, as the data were generated by MinION.

Can you remind me an online website to share large sequence files?

On Thu, Mar 12, 2020 at 4:31 PM cjw85 notifications@github.com wrote:

@zhanxw https://github.com/zhanxw

Can you confirm that when running guppy you are consistently using the modified base model, so you are running something like:

guppy_basecaller \ --save_path

--input_path \ --compress_fastq --fast5_out \ --config dna_r9.4.1_450bps_modbases_dam-dcm-cpg_hac_prom.cfg

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/nanoporetech/medaka/issues/139#issuecomment-598427059, or unsubscribe https://github.com/notifications/unsubscribe-auth/AABGRCASC4G5CGZQILRITITRHFIBTANCNFSM4K4ZH3QA .

[cjw85]

@zhanxw In order for medaka to make methylation predictions you will need to run the standalone guppy and use the modified base model as in the example command above.

[cjw85]

A warning has been added to the documentation to make the above point clear.

[TaniaJes]

Hi there,

When I try to run:

medaka consensus --save_features --check_output --model r941_min_high_g344 ./reads_minimap2.filtered.sorted.bam ./reads_minimap2.filtered.sorted.hdf

it seems like the program halts after reading the first few lines from the .bam file, and then fails to continue reading the bam file. (See error message below).

[22:25:18 - Predict] Setting tensorflow threads to 1. [22:25:18 - Predict] Processing 248460 long region(s) with batching. [22:25:18 - Predict] Using model: r941_min_high_g344_model.hdf5. [22:25:18 - ModelLoad] Building model with cudnn optimization: False [22:25:19 - DLoader] Initializing data loader [22:25:19 - Sampler] Initializing sampler for consensus of region ENST00000434970.2:0-9. [22:25:19 - Sampler] Initializing sampler for consensus of region ENST00000415118.1:0-8. [22:25:19 - Sampler] Initializing sampler for consensus of region ENST00000448914.1:0-13. [22:25:19 - Sampler] Initializing sampler for consensus of region ENST00000631435.1:0-12. Failed to read .bam file './reads_minimap2.filtered.sorted.bam'.%

How could I fix this?

Thanks, Tania

[cjw85]

@TaniaJes

I suspect that your reads_minimap2.filtered.sorted.bam file is either invalid or that you do not have a corresponding bam index (reads_minimap2.filtered.sorted.bam.bai) file alongside your bam. If you have further questions please start a new issue.