The result database does not contain metadata

[ynren1020]

Describe the bug Hello, when I run the command to get the output by using db = SampCompDB ( db_fn = "/results/outSampComp.db", fasta_fn = "/reference/transcripts_gencode.v38.fa")

It raised an error message saying: raise NanocomporeError("The result database does not contain metadata") nanocompore.common.NanocomporeError: The result database does not contain metadata

ps. the transcripts fasta file was produced by gtf and genome fasta file by gffread: gffread -w transcripts.fa -g /path/to/genome.fa transcripts.gtf

I am wondering how I should do to extract modification info from the resulting database. Thanks a lot for your help!

Yanan

[tleonardi]

Hi, it looks like there's something wrong with outSampComp.db. How did you generate it? Can you provide the log files of sampComp?

[ynren1020]

Dear Tommaso,

Thanks a lot for your immediate response!

I followed the instructions of this web page of nanocompore ( https://nanocompore.rna.rocks/data_preparation/). Specifically, below are an example command I used for one of my samples:

1. basecalling guppy_basecaller_cpu -i ./E1/fast5 -s ./E1/guppy_output_f5 -c rna_r9.4.1_70bps_hac.cfg --calib_detect --num_callers 16 --cpu_threads_per_caller 8 --client_id 111 --reverse_sequence true --fast5_out true

2. mapping to transcriptome minimap2 -x map-ont -t 16 -a ./reference/transcripts_gencode.v38.fa ./E1/guppy_output_f5/pass/*.fastq > E1.sam samtools view E1.sam -bh -t ./reference/transcripts_gencode.v38.fa.fai -F 2324 | samtools sort -@ 8 -o E1.filt.sorted.bam samtools index E1.filt.sorted.bam

3. resquiggle nanopolish index -s ${refer}/guppy_output_f5/sequencing_summary_111.txt -d ${refer}/fast5 ${refer}/guppy_output_f5/E1.fastq

nanopolish eventalign --reads ${refer}/guppy_output_f5/E1.fastq --bam ${refer}/minimap_output/E1.filt.sorted.bam --genome ./reference/transcripts_gencode.v38.fa --print-read-names --scale-events --samples > ${refer}/E1_eventalign_reads_gpu.tsv -t 40

4. collapse nanocompore eventalign_collapse -t 60 -i ${refer}/E1_eventalign_reads_gpu.tsv -o ${refer}/E1_eventalign_reads_collapsed.tsv

5. compare nanocompore sampcomp
   --file_list1 ${refer}/C1/C1_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv,${refer}/C2/C2_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv
   --file_list2 ${refer}/E1/E1_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv,${refer}/E2/E2_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv
   --label1 ctrl
   --label2 KO
   --fasta ./reference/transcripts_gencode.v38.fa
   --outpath ./results

ps. transcripts_gencode.v38.fa was made by this command: gffread -w /reference/transcripts_gencode.v38.fa -g /reference/hg38.fa /reference/gencode.v38.annotation.gtf

Attached please also find the log file from sampcomp step.

Thanks again for all your help, I appreciate your time and effort in helping me to figure out how to use Nanocompore.

Best,

Yanan

On Tue, Mar 15, 2022 at 4:44 AM Tommaso Leonardi @.***> wrote:

Hi, it looks like there's something wrong with outSampComp.db. How did you generate it? Can you provide the log files of sampComp?

— Reply to this email directly, view it on GitHub https://github.com/tleonardi/nanocompore/issues/196#issuecomment-1067766146, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGFIHCO6I3RARZTYZI7TX43VABLZDANCNFSM5QVUVO6A . Triage notifications on the go with GitHub Mobile for iOS https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Android https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub.

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[ynren1020]

Dear Tommaso,

I hope you are doing well. Just want to follow up with you on the issues I have got when I run nanocompore last two weeks. I am thinking if I have to re-run the analysis pipeline again by using transcriptome fasta file downloaded from Gencode website, rather than using the one I have generated by gffreads command. However, it would take a long time to re-run it again. But this is the only possible solution I can think of. Do you have any suggestions?

Thank you very much!

Yanan

Yanan Ren, Ph.D., M.S. Biostatistician The Hormel Institute University of Minnesota 801 16TH AVE NE, Austin, MN, 55912 @.*** | 770-688-0446

On Tue, Mar 15, 2022 at 9:22 AM Yanan Ren @.***> wrote:

Dear Tommaso,

Thanks a lot for your immediate response!

I followed the instructions of this web page of nanocompore ( https://nanocompore.rna.rocks/data_preparation/). Specifically, below are an example command I used for one of my samples:

1. basecalling guppy_basecaller_cpu -i ./E1/fast5 -s ./E1/guppy_output_f5 -c rna_r9.4.1_70bps_hac.cfg --calib_detect --num_callers 16 --cpu_threads_per_caller 8 --client_id 111 --reverse_sequence true --fast5_out true

2. mapping to transcriptome minimap2 -x map-ont -t 16 -a ./reference/transcripts_gencode.v38.fa ./E1/guppy_output_f5/pass/*.fastq > E1.sam samtools view E1.sam -bh -t ./reference/transcripts_gencode.v38.fa.fai -F 2324 | samtools sort -@ 8 -o E1.filt.sorted.bam samtools index E1.filt.sorted.bam

3. resquiggle nanopolish index -s ${refer}/guppy_output_f5/sequencing_summary_111.txt -d ${refer}/fast5 ${refer}/guppy_output_f5/E1.fastq

nanopolish eventalign --reads ${refer}/guppy_output_f5/E1.fastq --bam ${refer}/minimap_output/E1.filt.sorted.bam --genome ./reference/transcripts_gencode.v38.fa --print-read-names --scale-events --samples > ${refer}/E1_eventalign_reads_gpu.tsv -t 40

4. collapse nanocompore eventalign_collapse -t 60 -i ${refer}/E1_eventalign_reads_gpu.tsv -o ${refer}/E1_eventalign_reads_collapsed.tsv

5. compare nanocompore sampcomp
   --file_list1 ${refer}/C1/C1_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv,${refer}/C2/C2_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv
   --file_list2 ${refer}/E1/E1_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv,${refer}/E2/E2_eventalign_reads_collapsed.tsv/out_eventalign_collapse.tsv
   --label1 ctrl
   --label2 KO
   --fasta ./reference/transcripts_gencode.v38.fa
   --outpath ./results

ps. transcripts_gencode.v38.fa was made by this command: gffread -w /reference/transcripts_gencode.v38.fa -g /reference/hg38.fa /reference/gencode.v38.annotation.gtf

Attached please also find the log file from sampcomp step.

Thanks again for all your help, I appreciate your time and effort in helping me to figure out how to use Nanocompore.

Best,

Yanan

Yanan Ren, Ph.D., M.S. Biostatistician The Hormel Institute University of Minnesota 801 16TH AVE NE, Austin, MN, 55912 @.*** | 770-688-0446

On Tue, Mar 15, 2022 at 4:44 AM Tommaso Leonardi @.***> wrote:

Hi, it looks like there's something wrong with outSampComp.db. How did you generate it? Can you provide the log files of sampComp?

— Reply to this email directly, view it on GitHub https://github.com/tleonardi/nanocompore/issues/196#issuecomment-1067766146, or unsubscribe https://github.com/notifications/unsubscribe-auth/AGFIHCO6I3RARZTYZI7TX43VABLZDANCNFSM5QVUVO6A . Triage notifications on the go with GitHub Mobile for iOS https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Android https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub.

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