Squig pileup region error

[katiestoker]

Hello,

I am attempting to generate a signal to reference pileup using the following command:

REF=../linear_amplicon.fasta ALIGNMENT=02_MHhal_squig_align.bam OUTPUT_DIR=02_pileup REGION=pSHNJXXXLinear-_pOSIP:34-110

squigualiser plot_pileup -f ${REF} -s ../signals.blow5 -a ${ALIGNMENT} -o ${OUTPUT_DIR} --region ${REGION} --tag_name "pileup"

The pileup is successfully generated when I use any region between 1-30, however anything over 30 and I receive an error message (attached at the bottom) e.g. 1-31, 30-60 etc.

I receive the same error message when running the command on my other data set too...

Many thanks, Katie.

[hiruna72]

Hi @katiestoker,

Can you do a quick test separately by downloading the prebuilt executable (linux or macos) and running it on the given sample dataset?

The commands are available here

Thank you!

[katiestoker]

Hello,

I gave it a go and everything worked fine on the executable with the test data... I tried running my own data here too but received a key error message again:

Thanks!

[hiruna72]

Hello,

Really sorry for the trouble. Can you please upload a minimal dataset with the commands you ran so that I can reproduce the error? It makes it easy for me to debug.

Thank you.

[katiestoker]

Hello, Yes I have attached the files below... BAM file: 02_MHhal_squig_align.zip

Reference: linear_amplicon.zip

Blow5 subset: https://drive.google.com/file/d/1mJnZWlJ1LW87LR98np6s72ESMgavfxj-/view?usp=sharing

The commands I ran are as follows: REF=linear_amplicon.fasta

MAP_SAM=mapped_02_MHhal.sam mapped_02_MHhaI.zip

FASTQ=02_MHhal_filtered.fastq 02_MHhaI_filtered.zip

minimap2 -ax map-ont ${REF} ${FASTQ} -t8 --secondary=no -o ${MAP_SAM}

SIGNAL=signals.blow5 (the full dataset merged blow5 file) f5c index ${FASTQ} --slow5 ${SIGNAL}

ALIGNMENT=02_MHhal_squig_align.bam f5c eventalign -b mapped_02_MHhal.bam -r ${FASTQ} -g ${REF} --slow5 ${SIGNAL} -a -o 02_squig_eventalign.sam

samtools sort 02_squig_eventalign.sam -o ${ALIGNMENT} samtools index ${ALIGNMENT}

squigualiser plot_pileup -f ${REF} -s ${SIGNAL_FILE} -a ${ALIGNMENT} -o ${OUTPUT_DIR} --region ${REGION} --tag_name "pileup"

Apologies for the long comment!

Thanks for the help. Katie.

[hiruna72]

Hi Katie,

The issue is with the reference file I guess. It has both lower and upper case bases. When I replaced all with upper case it worked. Is there a valid reason to have a mix of cases?

[katiestoker]

Hello,

After changing this it has worked! I seem to have checked for this issue in the SAM file rather than the reference file... This is a mistake in the formatting, no valid reason for mix of cases.

Thanks for all your help.

All the best, Katie.

[hiruna72]

Hello,

Anyway, thank you for reporting this bug. I will update the dev branch to support bases regardless the case.

Also FYI, I have some pipeline scripts that have different plot types which I think might be useful to you.

[katiestoker]

Great!

I will take a look at the pipeline scripts too!

Thanks again for the help! (: