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cancerit / NanoSeq

Analysis software for Nanorate Sequencing (NanoSeq) experiments
GNU Affero General Public License v3.0
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0 reference calls error #101

Closed Jucarvag closed 3 months ago

Jucarvag commented 3 months ago

Hi, I am doing Nano-seq with human cells for the first time (I have been doing it with other species and it it has worked fine) and I am getting this error in the nanoseq_results_plotter.R: "ValueError: 0 reference calls. Exiting". Do you have any ideas why this would happen? I have tried with both GRCh37 and 38 (with their respective SNP and NOISE files). For what I can see, the steps previous to running runNanoSeq.py are working well. Thanks very much

fa8sanger commented 3 months ago

Hi, it’s difficult to know from here. It could be that there is very low duplicate rate, then the duplex consensus is never reached. But 0 reference calls is really too few. Have you checked the duplicate rate?

Another factor that may give 0 reference calls is when you use as matched normal a sample with very low coverage, always below the threshold.

Have a look at those things and let me know

On 29 Jul 2024, at 15:23, Jucarvag @.***> wrote:

Hi, I am doing Nano-seq with human cells for the first time (I have been doing it with other species and it it has worked fine) and I am getting this error in the nanoseq_results_plotter.R: "ValueError: 0 reference calls. Exiting". Do you have any ideas why this would happen? I have tried with both GRCh37 and 38 (with their respective SNP and NOISE files). For what I can see, the steps previous to running runNanoSeq.py are working well. Thanks very much

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Jucarvag commented 3 months ago

Thanks for responding so quickly! My duplication rate (calculated with efficiency_nanoseq.pl) is 72%. Filtered reads: 4x10^7 Neat reads: 1X10^7 I ran the efficiency on the normals, Filtered-normal: 2.2x10^6 Neat-normal: 1.8x10^6

fa8sanger commented 3 months ago

Do you only have 2.2 million reads in the matched normal? That’s super low coverage. That must be why you didn’t get any call

From: Jucarvag @.***> Sent: 29 July 2024 17:01:47 To: cancerit/NanoSeq Cc: Federico Abascal; Comment Subject: Re: [cancerit/NanoSeq] 0 reference calls error (Issue #101) [EXT]

Thanks for responding so quickly! My duplication rate (calculated with efficiency_nanoseq.pl) is 72%. Filtered reads: 4x10^7 Neat reads: 1X10^7 I ran the efficiency on the normals, Filtered-normal: 2.2x10^6 Neat-normal: 1.8x10^6

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Jucarvag commented 3 months ago

That's interesting. For my normals, I have always aimed to have 5%-10% of the reads I have for my nanoseqs. I guess that works well for smaller genomes but not so much for human cells. Should I aim to have the same number of reads in both normals and nanoseqs?

fa8sanger commented 3 months ago

For a good matched normal you need at least 10x which later translates in higher coverage because Nanoseq only sees part of the genome (restriction enzyme digestion).

From: Jucarvag @.***> Sent: 29 July 2024 17:15:18 To: cancerit/NanoSeq Cc: Federico Abascal; Comment Subject: Re: [cancerit/NanoSeq] 0 reference calls error (Issue #101) [EXT]

That's interesting. For my normals, I have always aimed to have 5%-10% of the reads I have for my nanoseqs. I guess that works well for smaller genomes but not so much for human cells. Should I aim to have the same number of reads in both normals and nanoseqs?

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Jucarvag commented 3 months ago

Perfect, thanks very much!